CA3140580A1

CA3140580A1 - Multivalent fzd and wnt binding molecules and uses thereof

Info

Publication number: CA3140580A1
Application number: CA3140580A
Authority: CA
Inventors: Stephane ANGERS; Sachdev Sidhu; Yuyong TAO
Original assignee: Antlera Therapeutics Inc
Current assignee: Antlera Therapeutics Inc
Priority date: 2019-06-11
Filing date: 2020-06-10
Publication date: 2020-12-17
Also published as: IL288730A; BR112021024787A2; EP3983443A1; US20220315659A1; AU2020291208A1; KR20220024460A; JP7377288B2; JP2022536142A; WO2020250156A1; CN114423784A; MX2021015439A; EP3983443A4

Abstract

Described herein are methods to affect binding by a multivalent binding molecule to a FZD receptor and a Wnt co-receptor on a cell wherein binding by the multivalent binding molecule to both FZD receptor and co-receptor on the cell activates a Wnt signaling pathway. Also described herein are multivalent binding molecules comprising a FZD receptor binding domain and a Wnt co-receptor biding domain on either end of an Fc domain that activate a Wnt signaling pathway and methods for their use.

Description

MULTIVALENT FZD AND WNT BINDING MOLECULES AND USES
THEREOF
BACKGROUND
100011 This Application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional application no. 62/860,161 filed June 11, 2019, the entirety of which is incorporated herein by reference.
100021 The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 10, 2020, is named 115773 PC424W0 SL.txt and is 279,203 bytes in size.
100031 Wnt signaling pathways are critical for embryonic development and tissue homeostasis in adults. Wnt ligands are secreted growth factors that regulate various cellular processes such as proliferation, differentiation, survival and migration. Wnt ligands are universally important for the control of tissue stem cells self-renewal and regulation of many progenitor cell populations. The hydrophobicity and sensitive tertiary structure of Wnt proteins have rendered their biochemical purification challenging and their use in vitro and in vivo impracticable.
100041 Nineteen Wnt ligands exist in humans that interact with a network of ten Frizzled cell surface receptors (FZD) and one of several co-receptors that guide the selective engagement of different intracellular signaling branches (Wodarz, A. and Nusse, R. Annu.
Rev. Cell Dev. Biol. 14, 59-88 (1998); Angers, S and Moon, R.T., transduction.
Nat. Rev.
Mot. Cell Biol. 10 , 468-477 (2009)). FZDs have conserved structural features including seven hydrophobic transmembrane domains and a cysteine-rich ligand-binding domain.
FZDs are known to function in three distinct signaling pathways, known as the Wnt planar cell polarity (PCP) pathway, the canonical Wnt/13-catenin pathway, and the Wnt/calcium pathway. Activation of Wnt signaling pathways also require the presence of Wnt co-receptors to dictate the differential engagement of intracellular signaling cascades regulating the expression of genes effecting cellular machineries underlying the cellular processes listed above. For example, Wnt ligands bind to a Frizzled receptor and a member of the low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co- receptor family to activate the Wnt/B-catenin pathway, or with a receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1/2), related to receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) co-receptor to initiate the Wnt/PCP pathway or alternate fIcatenin-independent signaling pathways. The Wnt/R-catenin pathway, sometimes referred to as the canonical pathway, culminates in the post-translational accumulation of the transcriptional effector R-catenin that interacts with T-cell factor/lymphoid enhancer factor (LEF/TCF) family of transcription factors to regulate the expression of context-specific genes.
SUMMARY OF THE INVENTION
100051 Wnts require lipidation for function (Janda et al., Science. 337, 59-64 (2012);
Kadowalci et al,, Genes Dev, 10, 3116-3128 (1996)) and their hydrophobic nature complicates biochemical manipulation; consequently, only a few Wnts have been purified (Willert et at., Nature 423, 448-452 (2003), Furthermore, Wnts are inherently cross-reactive for multiple receptors, especially when overexpressed or applied at high dose (He et al.
Science. 275, 1652-1654 (1997); Andres et al. Systematic mapping of Wnt-Frizzled interactions reveals functional selectivity by distinct Wnt-Frizzled pairs.
Journal of Biological (2015) (available at http://wvvw.jbc.org/content/ early/2015 /01/20/jbc.M114.
12648.short ).;
Holmen et al., J. Biol. Chem. 277, 34727-34735 (2002).). As a result, it has been impossible to activate Frizzled receptor complexes selectively to determine the specific functions of each in different contexts or to evaluate their therapeutic potential for degenerative conditions. The multivalent binding molecules and methods described herein activate preselected Frizzled receptor-coreceptor complexes selectively. Administration of the multivalent binding molecules described herein are contemplated to treat degenerative conditions by activating the appropriate Frizzled co-receptor complexes.
100061 Described herein are methods to affect binding by a peptide to a FZD receptor and a Wnt co-receptor on a cell wherein binding by the peptide to both FZD
receptor and the co-receptor activates a Wnt signaling pathway.
100071 Also described herein are multivalent binding molecules that activate a Wnt signaling pathway and methods for their use. The multivalent binding molecules bind to both an FZD receptor and a Wnt co-receptor thereby activating a Wnt signaling pathway. The multivalent binding molecules of this invention are also referred herein as "FZD agonists" or "FZDag". In a particular embodiment wherein the molecules of this invention bind a FZD
and a LRP5/6 the molecules maybe referred to as "Frizzled and LRP5/6 Agonists"
or "FLAgs". The multivalent binding molecules comprise an Fc domain, or fragment thereof comprising the CH3 domain, and a first binding domain that binds a FZD
receptor and a second binding domain that binds a Wnt co-receptor wherein the FZD binding domain is linked to one terminus of the Fc domain and the co-receptor binding domain is linked to the

2 other terminus of the Fe domain. Thus, the binding domain for the FZD receptor and the binding domain for the co-receptor are not directly linked rather they are separated by the Fe domain, or fragment thereof comprising the CH3 domain. This configuration of binding domains produces an unexpectedly high level of Wnt signaling pathway activation. The FZD
binding domain may be monovalent, having a single binding site (paratope) for a FZD
receptor, or may be multivalent having more than one binding site for a FZD
receptor, e.g., the binding domain may be bivalent, trivalent or tetravalent. The Wnt co-receptor binding domain may be monovalent, having a single binding site (paratope) for a Wnt co-receptor, or may be multivalent having more than one binding site for a Wnt co-receptor, e g., the binding domain may be bivalent, trivalent or tetravalent_ [0008] The methods described herein for producing the multivalent binding molecules enable selective and robust activation of any FZD receptor complex in vitro and an vivo.
Leveraging a panel of hundreds of synthetic antibodies targeting FZDs and their co-receptors, we generated multivalent binding molecules for selective and rational activation of one, two or multiple FZD receptors. The multivalent binding molecules of this invention are highly stable, amenable to large-scale production and facile purification, have predictable pharmacokinetics, and are contemplated to exhibit low immunogenicity.
[0009] In an embodiment of this invention the binding domains of the multivalent binding molecules described herein bind to one or more FZD receptors and an LRP, e.g., LRP 5 and/or LRP6 and are alternatively referred to herein as FLAgs. FLAgs that target particular FZDs and their LRP co-receptors will improve directed differentiation and cell therapy, sustain tissue organoid growth, and mobilize endogenous stem cells in vivo and promote tissue repair after injury and restore function following tissue degeneration.
[0010] The Fe domain of the FZD agonists may be an Fe domain of an immunoglobulin.
The immunoglobulin may be an IgG, e.g., an IgGit. In an embodiment of this invention the multivalent binding molecule is a peptide dimer wherein the peptides are dimerized via the intrinsic ability of Fe domain to dimerize or via a knob-in-holes configuration within the Fc which allows for specific assembly of two different peptides to produce multivalent binding domains. Methods for dimerizing peptides via a knob-in-hole configuration are described in W02018/026942, inventors Van Dyk et al. incorporated herein by reference, [0011] One or both of the multivalent binding domains of the FZD agonists described herein may be bivalent and monospecific, having two binding sites for the same epitope of their respective receptor or co-receptor targets. One or both of binding domains may be

3 bivalent and bispecific having two binding sites with each site binding a different epitope on their respective targets.
100121 In an embodiment of this invention the FZD binding domain may comprise two single chain variable fragments (scFv) for binding to the same or different epitopes on the FZD receptor. In other embodiments of this invention the FZD binding domain comprises one or more heavy-chain variable domain (VH) fragments and/or one or more light-chain variable domain (VL) fragments that bind the FZD. In other embodiments of this invention the FZD binding domain consists of one or more single-domain antibody fragments that bind to FZD. In other embodiments of this invention the FZD binding domain comprises a FZD
ligand or fragment thereof that binds the FZD receptor. In an embodiment of this invention the FZD binding domain comprises a synthetic peptide that binds the FZD, e.g., an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin. In an embodiment of this invention the FZD multivalent binding domain does not comprise scFv.
The FZD ligand may be, e.g., a fragment of Wnt protein or of Norrin that binds the FZD
receptor, or another natural or synthetic peptide that is affinity matured to interact with one or more FZD receptors. Norrin is a FZD4-specific ligand that, in complex with LRP5 and/or LRP6, is associated with activation of canonical Wnt signaling.
100131 In an embodiment of this invention, the co-receptor binding domain may comprise two single chain variable fragments (scFv) for binding to the same or different epitopes on the co-receptor. In other embodiments of this invention the Wnt co-receptor binding domain comprises one or more heavy-chain variable domain (VH) fragments and/or one or more light-chain variable domain (VL) fragments that bind the Wnt co-receptor. In other embodiments of this invention the co-receptor binding domain consists of one or more single-domain antibody fragments that bind to the co-receptor. In an embodiment of this invention the Wnt co-receptor binding domain comprises a peptide that binds the Wnt co-receptor wherein the peptide is a fragment of a naturally occurring ligand that binds the Wnt co-receptor or is a synthetic peptide that binds the Wnt co-receptor, e.g., an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin. In another embodiment of this invention the co-receptor binding domain comprises a co-receptor ligand or fragment thereof that binds the co-receptor (for example the ligand Dkkl for the co-receptor LRP5/6) or another natural or synthetic peptide affinity matured to interact with one or more co-receptors.
100141 In an embodiment of this invention the co-receptor multivalent binding domains

4 do not comprise scFv.
100151 In an embodiment of this invention each binding domain of the molecules described herein may be formed by two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair with the VL and VH of the other peptide forming a diabody. In this configuration, the binding domain has two binding sites that bind to its target, i.e., the FZD binding domain has two binding sites for the FZD receptor and the co-receptor binding domain has two binding sites for the co-receptor. Using a knobs-in-holes Fc configuration, the peptides comprising the VH and VL can be engineered such that they are non-identical but still pair to form a bispecific binding domain capable of binding to two different sites on the FZD receptor or co-receptor (see Figure 3A).
100161 In an embodiment of this invention one or both of the multivalent binding domains comprise two peptides forming a diabody on each terminus of the Fc domain. Each diabody has two binding sites for an epitope on their respective FZD receptor or co-receptor targets. The diabody may be monospecific wherein the binding sites bind the same epitope on the FZD receptor or co-receptor, or the diabody may be bispecific binding to two different epitopes on the FZD receptor or co-receptor.
100171 The peptides forming the scFv or diabodies may be derived from an antibody that binds to a FZD receptor or from an antibody that binds to a Wnt co-receptor.
For the FZD
binding domain, the antibody may be an antibody that binds to one or more FZD
receptors and antagonizes Wnt signaling or inhibits Wnt binding to given FZD
receptor(s), or the antibody may be an antibody that binds to one or more FZD receptors without inhibiting Wnt binding to the FZD receptor. For the co-receptor binding domain, the antibody may be an antibody that binds to the co-receptor and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor or the antibody may be an antibody that binds to a co-receptor without inhibiting Wnt binding to the co-receptor.
100181 The FZD binding domain may bind to one or more members of the FZD receptor family, e.g., Frizzled Class Receptor 1 (FZD1), Frizzled Class Receptor 2(FZD2), Frizzled Class Receptor 3 (FZD3), Frizzled Class Receptor 4 (FZD4), Frizzled Class Receptor 5 (FZD5), Frizzled Class Receptor 6 (FZD6), Frizzled Class Receptor & (FZD7), Frizzled Class Receptor 1 Frizzled Class Receptor 8 (FZD8), Frizzled Class Receptor 9 (FZD9), or Frizzled Class Receptor 10 (FZD10). The co-receptor binding domain may bind to any Wnt co-receptor, e.g., LRP5/6, PTK7, ROR1/2, RYK, GPR124, TSPAN12, or CD133. In an embodiment of this invention the co-receptor binding domain binds to LRP5 and/or LRP6.
In an embodiment of this invention the co-receptor binding domain binds to a single epitope on a co-receptor, e.g., an epitope of the LRP protein that binds Wntl or Wnt3a. In an embodiment of this invention the co-receptor binding domain binds to two epitopes on a co-receptor, e.g., an epitope on an LRP that binds to Wntl and an epitope that binds to Wnt3a.
100191 An embodiment of this invention includes methods for producing induced pluripotent stem (iPS) cells comprising culturing a somatic cell under conditions suitable for reprogramming the somatic cells in the presence of an effective amount of a multivalent binding molecule described herein. The multivalent binding molecule may be included in an amount to accelerate the generation of iPS cells as compared to the generation of iPS cells in the same culture conditions without the multivalent binding molecule.
100201 Also an embodiment of this invention are methods for directing differentiation of iPS or other pluripotent stem cells (PSCs) towards various lineages by culturing these cells in the presence of an effective amount of a multivalent binding molecule described herein.
10021] An embodiment of this invention includes methods for generating tissue organoids comprising culturing a tissue sample under conditions suitable for the generation of organoids in the presence of an effective amount of a multivalent binding molecule described herein as part of the culture cocktail. In an embodiment or this invention, the frequency of generating tissue organoids cultured in a medium comprising the multivalent binding molecule is enhanced as compared to organoids cultured in the same medium without the multivalent binding molecule. In an embodiment or this invention, the tissue organoids are generated more rapidly when cultured in a medium comprising the multivalent binding molecules as compared to tissue samples cultured in the same medium without the multivalent binding molecules.
An embodiment of this invention includes methods for enhancing the maintenance of tissue organoids comprising culturing an organoid in the presence of an effective amount of a multivalent binding molecule described herein as part of the culture cocktail.
As described herein, the survival of tissue organoids cultured in a medium comprising the multivalent binding molecules is prolonged as compared to organoids cultured in the same medium without the multivalent binding molecule.
[0022] An aspect of this invention is a method for making the multivalent binding molecules described herein. In an embodiment of this invention the multivalent binding molecule is generated by, a) selecting an Fc domain having a C-terminus and an N-terminus, b) identifying an antibody that binds to one or more FZD receptors and c) identifying an antibody that binds to one or more Wnt co-receptors, d) generating a nucleic acid molecule comprising a nucleotide sequence that encodes (i) the Fc domain of step a, (ii) a nucleotide sequence that encodes a VL and/or a VH of the antibody of step b, or a 'IL and/or a VH derived from the antibody of step b that binds the one or more FZD, and (iii) a nucleotide sequence that encodes a 'IL and a NTH of the antibody of step c, or a 'IL and a VH derived from the antibody of step c that binds to the one or more Wnt receptors of step c, e) expressing the nucleic acid molecule of (d) to produce a polypeptide wherein the polypeptide dimerizes to form a multivalent binding molecule comprising an Fc domain, a FZD binding domain and a Wnt co-receptor binding domain wherein, the FZD binding domain comprises of the VL and VH of the antibody of step b or derived from the antibody of step b and is linked to one terminus of the Fc domain, and the Wnt co-receptor binding domain comprises the 'IL and VH of the antibody of step c or derived from the antibody of step c and is linked to the other terminus of the Fc domain thereby forming the multi-specific binding molecule.
The antibody in step (b) may be an antibody or antibody fragment that binds to one or more FZD receptors and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. The antibody in step (b) may be an antibody or antibody fragment that binds to one or more FZD
receptors without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. The antibody in step (c) may be an antibody or antibody fragment that binds to one or more of the Wnt co-receptors and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor, or binds to the co-receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the co-receptor. The binding domains may be linked to the Fc domain via a linker. The modular aspects of this invention allows for mixing and matching binding domains of antibodies for any given FZD receptor and co-receptor on the termini of the Fc domain to generate a multivalent binding molecule that can engage multiple Frizzled receptor - co-receptor complexes or to selectively engage a single Frizzled receptor-co-receptor complex to activate Wnt signaling.

[0023] The multivalent binding molecule comprises a peptide dimer configured to have an Fc domain and a binding domain that binds one or more FZD receptors and a second binding domain that binds one or more Wnt co-receptors wherein the FZD binding domain is linked to one terminus of the Fc and the co-receptor binding domain is linked to the other terminus of the Fc Each binding domain may be monovalent or multivalent, e.g.
bivalent, trivalent or tetravalent.
[0024] Also an embodiment of this invention are methods using the multivalent binding molecules, e.g., for producing induced pluripotent stem (iPS) cells, for directed differentiation of pluripotent stem cells, and for generating and/or maintaining tissue organoids, or to enhance tissue regeneration in a subject in need thereof.
[0025] Additional embodiments of this invention are methods for activating Wnt signaling pathways for the mobilization of endogenous stem/progenitor cell pools for regenerative medicine and for disorders or diseases associated with insufficient Wnt signaling.
BRIEF DESCRIPTION OF THE FIGURES
[0026] Figure lA depicts the binding specificity of five antibodies selected for their binding to the extracellular domain (ECD) of human LRP6. LRP6-binding antibodies were selected from a synthetic antibody library by selecting for antibodies that bound the recombinant extracellular domain (ECD) of human LRP6. The antibodies were assayed by ELISA for binding to human LRP6, mouse LRP6, and mouse LRP5. Binding to an Fc peptide and bovine serum albumin (BSA) were included as negative controls.
[0027] Figure 1B depicts the results of a luciferase reporter assay monitoring Wnt signaling activation demonstrating that IgG 2539 and IgG 2542 (1000/1) bind different sites on LRP6 ECD by their opposite effects on Wntl (Transient transfection) and Wnt3a (0.5 pig/m1 purified protein) stimulation. Anti-MEP antibody acts as control.
[0028] Figure 2A depicts a representative bispecific IgGs (Bi-IgG) and bispecific diabody (bi-diabody) comprising of a FZD binding domain (5019) and an LRP6-W1 (2942, L61) or -W3 (2539, L63) binding domains on the same end of the Fc domain [0029] Figure 2B demonstrates that the bispecific IgGs (5019-2539 Bi-IgG and 5019-2542 Bi-IgG) do not activate Wnt signaling but rather act as antagonists of Wnt signaling, as determined in a TOPFlash luciferase reporter assay in HEK.293 cells.
[0030] Figure 2C -2G depict the binding of bispecific diabodies wherein the Fc domains are in a knob/hole configuration (K/H). Two resultant diabodies 5019-2539-K/H
(FZD/L1tP6-W3) and 5019-2542-K/H (FZD/LRP6-W1) retain the FZD binding profile of the original IgG as well as the LRP6 binding activity though very weak. FIG. 2C
depicts the purified FZD-LRP6 diabodies: 5019-2539-K/H and 5019-2542-IC/H. FIG. 2D depicts the FZD receptor binding profile of the 5019-diabody to FZD4, FZD5, and FZD7. The IgG was previously characterized to bind to FZD1, 2, 4, 5, 7, 8. FIG. 2E
depicts the FZD
receptor binding profile of the bi-specific FZD/LRP6 diabody 5019-2539- KJH.
FIG. 2F
depicts the FZD receptor binding profile of the bi-specific FZD/LRP6 diabody KM. FIG. 2G demonstrates that the homo (2539-Fc and 2542-Pc) and hetero-diabodies (5019-2539-Fc and 5019-2542-Fc) having the binding domains on one terminus of the Pc domain interact with the LRP6 extra-cellular domain. Figure 2H demonstrates co-binding of the diabodies 5019-2539-KJII and 5019-2542-KM to FZD CRD and LRP6 ECD in solution as determined in Bio-Layer Interferometry (BLI) assays.
[0031] Figure 21 demonstrates neither 5019-2539-K/11 or 5019-2542-K/H, wherein the FZD and LRP6 receptors diabodies forming the binding domains are present on the same side of the Fc, are FZD agonists that activate a Wnt mediated pathways. The results demonstrate the 5019-2539-K/H diabody (selective for the Wnt3 site on LRP6) completely blocks the Wnt3-mediated pathway activation at lOtiM and 50nM whereas the 5019-2542-K/H
is less effective as revealed using the TOPFlash luciferase reporter assay in 11E1(293 cells.
[0032] Figure 2J depicts a comparison of the luciferase activity of a tetravalent binding molecule having binding domains comprising diabodies or scFvs. The molecules having binding domains comprising anti-FZD scFvs and anti-LRP diabodies (Fr+r-L61+3) exhibited similar activity to the molecules having binding domains comprising anti-FZD
diabody and anti-LRP diabodies (F"-L61+3). In contrast, as compared to the F"-L6' t3 molecules activity was significantly reduced for the molecules that contained anti-FZD diabodies but anti-LRP6 scFvs (FP P-L61* 3t) or scFvs at both ends (FN N-L6P 3*).
[0033] Figure 2K and Figure 2L demonstrate the differences in activity between a tetravalent binding molecule having binding domains comprising diabodies or scFvs were not due to differences in affinity, as BLI measurements showed comparable, high-affinity binding to LRP6 and FZD isoforms regardless of whether paratopes were presented in the diabody or say format.
[0034] Figure 3A a schematic representation of a tetravalent binding molecule wherein two FZD binding domains comprised of homo (recognizing the same epitope) or hetero (recognizing separate epitopes) diabodies are linked to one end of an Fc domain and two LRP6 binding domains comprised of homo or hetero diabodies are linked to the other end of the Fc domain.
100351 Figure 3B depicts binding by the multivalent binding molecule 5019-Fc-2539 (FP+P-L63+3). and 5019-Fc-2542 (FP+P-L61+1). to FZD4, FZD5 and FZD7 ECDs.
Binding to FZD receptors is detected using BLI assays.
100361 Figure 3C depicts activation of the Wnt-lIcatenin signaling pathway by tetravalent binding molecules 5019-Fc-2539 (FP+P-L63F3)., 5019-Fc-2542 (FP+P-L61+1)., 5019-2542 (FP1-P-L61+3), and purified Wnt3A (0.5 g/m1). The concentration of the molecules is indicated. The tetravalent binding molecules are agonists that robustly activate the Wnt-13catenin pathway in HEK293T cells as measured using the pBAR luciferase reporter assay.
The 5019-Fc-2539 homodiabody binds to multiple FZD receptors (5019: FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site on LRP6 (2539) and activates the reporter to levels comparable to purified Wnt ligands. The 5019-KM-2539:2542 heterodiabody, which binds to both Wnt binding sites on LRP6 is more effective.
100371 Figure 3D depicts Wnt-ficatenin pathway activation by multivalent binding molecules having a FZD homodiabody (5019) linked through the Fc to either monospeciftc LRP6 homodiabody (5019-Fc-2539, 5019-Fc-2542) or bispecfic LRP6 heterodiabody (5019-KM-2539-2542, also known as 5019Ag or FP+P-L61+3).
100381 Figure 3E depicts the activation of Wnt-fIcatenin signaling by molecules comprising a monovalent binding domain for either the FZD receptor or the LRP6 co-receptor. Wnt-lIcatenin pathway activation was detected using pBAR luciferase reporter assays performed in HEK293T cells. 5019-MBP-KM-2539-2542 contains one monovalent binding domain for FZD and still activates the Wnt pathway, but showing an 8-fold decrease in efficacy with respect to 5019Ag (which contains two FZD binding domains to the same epitope). 5019-KM-2539-MBP, which retains only one LRP6-W3 binding domain in the C-terminus, exhibits much less efficacy. Importantly, minimal agonistic activity was detected for the two mono-FZD:mono-LRP6 diabodies 5019-MBP-K/H-2539-MBP and 5019-MBP-IQH-MBP-2542 as well as the one LRP6-W1 site diabody 5019-KJH-MBP-2542.
100391 Figure 3F depicts Wnt-fIcatenin pathway activation by a tetravalent binding molecule in which an anti-LRP5 paratope targeting the WNT3A binding site was substituted for the anti-LRP6 paratope targeting the WNT1 binding site to generate a molecule (FP P-L5/63) that could recruit both co-receptors and observed activity similar to that of FP+P-L611-3 to (EC50 =4 nM), [0040] Figure 4A depicts Wnt-acatenin pathway activation in reporter cells without an endogenous FZD4 receptor (-FZD4) or modified to express the FZD4 receptor (+FZD4) by a multivalent binding molecules having FZD binding domains (homodiabodies in this case) specific for FZD4 on one side of the Fc domain and a co-receptor binding domain for LRP6 ( 2539 and 2542) on the other side of the Fc domain (FZD4Ag: 5038Ag/5038-K/H-2542, 5044Ag/5044-KM-2539-2542, 5048Ag/5048-KJH-2539-2542, 5063Ag/5063-KM-2539-2542, 5080Ag/50180-K/H-2539-2542, 5081Ag/5081-KJH-2539-2542). Controls are the multivalent binding molecule 5019Ag (5019-K/H-2539-2542), and Norrin, an endogenous agonist of FZD4. The results demonstrate that replacing the 5019 FZD binding domain (recognizing FZD1, 2, 4, 5, 7, 8) within the 5019Ag/5019-KM-2539:2542 (a pan-FZD agonist) with selective binding domains for FZD4, enabled the development of selective FZD4 agonists. HEK293T cells were transfected with pBARL (Wnt-(3catenin luciferase reporter) and Rluc (normalization control), plasmids coding for the listed FZD
agonists and with or without FZD4 and LRP6 cDNA. Norrin was used as a positive control for activation of FZD4. HEK293T cells express low to not detectable levels of FZD4 therefore agonists were only able to activate the reporter gene in the presence of transfected FZD4 cDNA. In contrasts, the pan- FZDag 5019-K/H-2539:2542 robustly activates Wnt-Pcatenin signaling in the absence or presence of FZD4 through activation of other endogenously expressed Frizzled in these cells.
[0041] Figure 4B depicts Wnt-Rcatenin pathway activation by multivalent binding molecules having binding domains (homodiabodies) specific for FZD2 (2876, 2890), FZD2/7 (2886) FZD6 (2747), or FZD9/10 (2969, 2974) on one side of the Fc and the LRP6 heterodiabody formed by 2539 and 2542 antibody fragments on the other side of the Fc.
Wnt-Rcatenin pathway activation was evaluated using the pBARL assay in HEK293T
cells.
[0042] Figure 4C depicts Wnt pathway activation by multivalent binding molecules having FZD binding domains that are pan-specific for FZD and derived from IgG
that block Wnt binding to FZD and Wnt-Rcatenin signaling. The LRP6 binding domains in these molecules are on the c-terminus of the Fe and consist of a diabody formed by antibody 2539 and 2542, which have paratopes recognizing the Wnt3 and Wntl binding sites on respectively.
[0043] Figure 4D depicts Wnt pathway activation by multivalent binding molecules having FZD binding domains that are pan-specific for FZD and derived from IgG
that do not block Wnt binding to FZD and do not antagonize Wnt3-induced pathway activation. The LRP6 binding domains in these molecules are on the c-terminus of the Fc and consists of a diabody formed by antibody 2539 and 2542, which have paratopes recognizing the Wnt3 and Wntl binding sites on LRP6 respectively.
100441 Figure 5 depicts a comparison of the FZD/LRP6 binding behavior of three tetravalent binding molecules of this invention. 5019-Fc-2539, 5019-Fc-2542, 5019-Fc-2539-2542 bind tightly to FZD but exhibit weaker LRP6 interaction (left graph) or FZD/LRP6 co-binding (middle graph). The FZD binding profile of 5019-K/H-2539-(right graph) shows it recognizes FZD4, FZD5 and FZD7.
100451 Figure 6A is an illustration of the top two propellers (E1-E2) of LRP5/6 known to mediate binding with Wntl, and binding of the bottom 2 propellers (E3-E4) of LRP5/6 that are proximal to the plasma membrane and known to mediate interaction with Wnt3. Figure 64 also illustrates Wntl interacting with LRP5/6 and the FZD receptor and Wnt3 interacting with LRP5/6 and the FZD receptor.
100461 Figure 6B is an illustration of a possible interaction of the FZD receptor and LRP5/6 receptor by the multivalent binding molecules 5019-Fc-2539, 5019-Fc-2542 and 5019-KM-2539-2542.
100471 Figure 6C demonstrates the multivalent binding molecules are agonists that robustly activate the Wnt-I3catenin pathway in HEK293T cells as measured using the pBAR
luciferase reporter assay. 5019-Fc-2539 homodiabody binds to multiple FZD
receptors (5019 binds FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site on LRP6 (2539) and activates the reporter to levels comparable to purified Wnt ligands. The 5019-K/H-2539:2542 heterodiabody, which binds to both Wnt3a and Wntl binding sites on LRP6, is more effective.
100481 Figure 6D demonstrates 5019-KM-2459:2460, a tetravalent binding molecule having an Fe domain in a knob-in-hole configuration and having a FZD binding domain (homodiabody) that is pan FZD-specific (5019) and a co-receptor binding domain that is bispecific (heterodiabody) for two sites on LRP5 (2459 binds Wntl binding site and 2460 binds Wnt3 binding site), also activates the Wnt43catenin pathway in HEK293T
cells.
100491 Figure 7A demonstrates that by replacing the FZD
binding domain within the 5019-K/H-2539:2542 (a pan-FZD agonist recognizing FZD I, 2, 4, 5, 7, 8) with a FZD
binding domain specific for FZD5 (#2928), a selective FZD5 agonist was generated. HPAF-II
cells have been shown to depend on FZD5 signaling for their proliferation.
Blocking Wnt-FZD5 signaling using the Wnt secretion inhibitor LGK974 (targeting the acyl-transferase Porcupine) leads to cell cycle arrest and inhibition of proliferation.
Proliferation can be rescued with addition of exogenous Wnt3a conditioned media or with the addition of the FZD5 selective agonists (2928-K/H- 2539:2542) or pan-FZD agonist (5019-K/H-2539:2542) described herein. The FZD4 selective agonist 5038-K/H- 2539:2542 only has modest rescue ability.
100501 Figure 7B demonstrates stimulation of C3H10T1/2 cells with a FZD2-specific FLag led to robust induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with a Pan-FZD FLAg, whereas a FZD5-specific FLAg exhibited minimal activity.
100511 Figure 8A and 8B demonstrates that the pan-FZDag (F-L6'3) of this invention fully substitute for exogenous Wnt3A conditioned media to rescue the growth inhibition of intestinal organoids when Wnt secretion is blocked with LGK974, a small molecule inhibitor of Porcupine (lower left photograph). Intestinal organoids isolated from mice grow in the presence of recombinant R-Spondin and require the presence of Wnt ligands secreted by the paneth cells. Figure 8A depicts inhibition of Wnt production using LGK974 leads to organoid death (upper right photograph). Exogenous application of Wnt3A conditioned media (lower light photograph) or FZDag (lower left photograph) rescues organoid growth in the presence of LGK974. The upper left photograph depicts organoids treated with DMSO
without LGK974 as control. Figure 8B demonstrates that inhibition of Wnt production by LGK974, leading to organoid death, can be rescued by application of Wnt3A conditioned media or FZDag (FP+P-L61+3), as quantified using CellTiter Glow Assay, Promega.
[0052] Figure 9A and 9B depict an example of the plasmids encoding the peptides that dimerize in a knob-into-hole conformation to form the pan-FZDag 5019-KH-2539-2542(FP+P-L61+3). Figure 9A depicts a plasmid encoding the peptide comprising an Fc region comprising a "knob" mutation, the VH and VL of panFZD antibody #5019, and the VL of LRP antibody #2542 and the VH of LRP antibody #2539. Figure 9B depicts a plasmid encoding the peptide comprising a nucleic acid encoding Fc region comprising a "hole"
mutation, the VH and VL of pan-FZD antibody #5019, and the VH of LRP antibody #2542 and the VL of LRP antibody #2539. The peptides encoded by these plasmids form a heterodimer having tetravalent binding domains comprising a homo-diabody produced by pairing of the VH and VL of the pan specific FZD antibody #5019 and a bispecific heterodiabody produced by the pairing of VL of LRP6 antibody #2539 and VH of LRP
antibody #2542 from one peptide with the VH of LRP antibody #2539 and the VL
of LRP

antibody #2542 of the other peptide.
100531 Figure 9C is a schematic representation of the heterodimer knob-into-hole configuration 5019-1C/11-2539:2542 (F1+P-L61+3). Using a knob-in-hole configuration within the Fc it is possible to increase the modularity of the molecule up to 4 different binding sites.
For this molecule (5019-K/H-2539:2542) a pan-FZD homodiabody is engineered on one side of the Fe domain and an heterodiabody containing Wnt3 (2539) and Wnt1 (2542) binding sites on the other side of the Fe domain 100541 Figure 10A and 10B is an annotation of the domains of the nucleic acid sequence of the 5019-knob-2539:2542 multivalent binding molecule (SEQ ID NO: 21 plus an additional 3' TGA, and its complementary sequence).
100551 Figure 11 A-F depict the design and validation of tetravalent binding molecules that bind FZD and LRP6 Wntl and Wnt3 binding sites (FLAgs) as activators of the Wnt-13catenin pathway. FIG. 11A depicts anti-FZD Fab inhibitory (top) and specific activity (bottom). FIG. 118 depicts inhibition of Wntl or Wnt3A signaling by the indicated LRP6 Abs in the diabody-Fc format. FIG. 11C depicts molecular architecture of tetravalent FLAgs.
FIG11D shows dose response curves for the activation of a LEF/TCF reporter gene (y-axis) in HEK293T cells by serial dilutions of pan-specific FLAg proteins (F P P -L61+1, F PIP -L63+3 and FP+P-L61+3) (x-axis). FIG. 11E depicts the levels of ficatenin protein in RICO cells after 30 min treatment with indicated concentrations of pan-FLAg (F P+P-L61+3), FIG.
11F depicts the time course of flcatenin and phosphorylated Dishevelled-2 (p-Dv12) protein levels in RKO
cells treated with 10 nIV1 pan-FLAg (FP+P-L6I+3).
100561 Figure 12A-Figure 12D depict the characterization and dissection of the FLAG
FP+P-L611-3 binding and activity. FIG. 12A and 12B depicts binding kinetics of FP-L6'1-3 to nine of 10 human FZD CRDs to human LRP6 ECD. FIG. 12C demonstrates F'-L63 behaved similarly to a conventional IgG and interacted with FcRn in a dose and pH
dependent manner. FIG. 12D demonstrates FP-P-L61t3 also behaved similarly to the IgG for interaction with other Fe effectors including complement (Clq), the natural killer cell marker CD16a, the B cell marker CD32a, and the monocyte and macrophage marker CD64.
00571 Figures 13A and 13B demonstrate that treatment of with 30 TIM F'-L&3 for three days caused robust induction of the mesoderm marker BRACHYURY and decreased expression of the pluripotency marker OCT4 to levels comparable to treatment with the GSK3 inhibitor CHIR99021 at 6 M.
100581 Figure 14 displays representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with vehicle, C59 or pan-FLAg(FP+P-L61 3 ) + C59.

is expressed in the stem cells at the bottom of crypts. Cell nuclei were counterstained with DAPI.
DETAILED DESCRIPTION OF THE INVENTION
100591 Described herein are multivalent binding molecules comprising an Fc domain, a FZD binding domain and a binding domain for a Wnt co-receptor wherein the binding domains are attached to opposite ends of the Fc domain. The multivalent binding molecules of this invention are agonists of a Wnt signaling pathway and are alternately referred to herein as FZD agonists or FZDag. Wnt ligands function by promoting the clustering of FZD
receptors with co- receptors. Without wishing to be bound by theory it is contemplated that the multispecific molecules described herein simultaneously bind to a FZD
receptor and a Wnt co-receptor and thereby activate Wnt signaling pathways.
100601 The modularity and effectiveness of the multivalent binding molecules for activating Wnt signaling pathways described herein contrasts with the Wnt surrogates described in the prior art which consists of monovalent FZD and LRP5/6 binding ligands, wherein the binding ligands are not attached to opposite ends of an Fc domain.
In an embodiment of this invention the FZD binding domain comprise a binding moiety that is derived from antibodies or polypeptides that bind specifically to one or more FZD receptors and the co-receptor binding domain comprises a binding moiety that binds to a co-receptor, e.g., an LRP5/6, ROR1/2, RYK or PTK7. In an embodiment of the invention the antibodies or polypeptides that specifically bind to one or more FZD receptors bind to a cysteine rich domain (CRD) of one or more of the FZD receptor.
100611 The amino acid sequences of FZD receptors and nucleotide sequences encoding FZD receptors, and antibodies and libraries of antibodies that bind FZD or the Wnt co-receptors LRP5/6, ROR1/2, RYK or PTK7 are readily available or can be generated using methods well known in the art (see e.g., U.S. publication no. 2015/0232554, inventors Gurney et al. and US publication no. 2016/0194394, inventors Sidhu et al. and US
20190040144, inventors Pan et al.; U.S. publication no. 2017/0166636, inventors Wu et al.;
U.S. publication no. 2016/0208018, inventors Chen et al.; U.S. publication no.
2016/0053022, inventors Macheda et al.; U.S. publication no. 2015/031293, inventors Damelin et al.).
100621 Methods for generating peptides or polypeptides that bind to a selected target are well known in the aft, see for example Sidhu et al. Methods in Enzymology (2000) 328: 333-336. For example, a library of affibodies that bind a FZD or Wnt co-receptor may be obtained according to protocols known in the art (see, e.g., U.S. Pat. No.

5,831,012 and Lofblom et al., FEBS Letters 584 (2010) 2670-2680); a library of ankyrin repeat proteins used for the selection of a peptide that binds a FZD or Wnt co-receptor may be obtained according to protocols known in the art (see e.g., WO 02/020565, inventors Stumpp et al.) and a library of fibronectin repeat proteins used for the selection of a peptide that binds a FZD or a Wnt co-receptor may also be obtained according to protocols known in the art (see e.g., U.S. Patent No. 9,200,273, inventors Diem and Jacobs. The peptides that bind to a FZD
or a Wnt co-receptors may also be fynotners, small binding proteins derived from the human Fyn SH3 domain or artificial receptor proteins, "anticalins", based on human apoliprotein D, and may be generated using methods known in the art, see e.g., Silacci et al., J. Biol. Chem (2014) 289(20):14392-8 and Vogt and Skein, ChemBioChem (2004) 5, 191-199) .
[0063] Antibodies suitable as the source for antigen binding peptides as described herein may be isolated by screening combinatorial libraries for polypeptides with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554;
Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003);
Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol, Biol.
340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132(2004), In certain phage display methods, repertoires of VET and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12:
725-734 (1993). Finally, naive libraries can also be made synthetically by cloning

6 unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mo1. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example:
U.S. Pat. No.
5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
[0064] Thus one of skill in the art would readily prepare an Fc domain and mix and match multivalent FZD binding domains and Wnt co-receptor binding domains having a desired specificity on the N and C terminals of the Fc domain to prepare the multivalent binding molecules to bind the desired FZD receptors and co-receptors and thereby activate specific Wnt pathways. These specific agonists would serve as powerful tools in enhancing cell proliferation, differentiation, organoid survival and maintenance, and tissue regeneration in vivo. These specific agonists also serve as powerful tools for profiling the FZD specificity involved in these processes. For example, as shown herein, the FZD5Ag but not FZD4Ag rescues the growth defect of L6K974-treated RNF43 mutant PDAC cell lines, highlighting the importance of FZD5 over FZD4 receptor in this process.
[0065] An embodiment of this invention is a method to effect binding by a peptide to a FZD receptor and a Wnt co-receptor on a cell wherein binding by said peptide to both FZD
receptor and co-receptor activates a Wnt signaling pathway in cell. The method comprises selecting an Fc domain, or fragment thereof comprising a CH3 domain, having a C-terminus and an N-terminus, linking a first multivalent binding domain that binds the FZD receptor on one terminus of the Fc domain, and linking a second multivalent binding domain that binds to the Wnt co-receptor on the other terminus of the Fc domain thereby forming a multivalent binding molecule and then contacting the multivalent binding molecule with a cell expressing said FZD receptor and co-receptor under conditions to activate the Wnt signaling pathway.
[0066] In an embodiment of the invention the multivalent binding domains may comprise single chain variable fragments (ScFv) that bind to one or more FZD receptor, a ligand of the FZD receptor or co-receptor, or a fragment thereof that binds to the FZD
receptor or the co-receptor. In another embodiment the binding domains do not comprise single chain variable fragments (ScFv) that bind to one or more FZD receptor, a ligand of the FZD
receptor or co-receptor, or a fragment thereof that binds to the FZD receptor or the co-receptor.

[0067] In an embodiment of the invention at least one of the FZD or co-receptor multivalent binding domain comprises a diabody having two peptides each peptide having a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL), wherein the VH and the VL from one peptide pairs with the VL and VH of the other peptide such that the binding domain has two epitope-binding sites. The VH and VL domains may be the VH and VL of an antibody that binds to a Wnt binding site on the FZD receptor or co-receptor. A
VH or VL derived from an antibody, the source antibody, may be 50%, 55%, 60%, 75%.
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH and VL of the source antibody and still retain binding to the FZD receptor or co-receptor site bound by the antibody.
[0068] In an embodiment of this invention the multivalent binding molecules of this invention comprise the multivalent binding molecules of Table 1 (Table 1 comprises Tables lA and 113: Table 1A indicates nucleotide sequences and amino acid sequences of exemplified multivalent binding molecules of this invention; Table 1B
indicates the nucleotide sequences encoding the various domains of the exemplified multivalent binding molecules). In an embodiment of this invention the multivalent binding molecules of this invention consist essentially of the multivalent binding molecules of Table 1.
In an embodiment of this invention the multivalent binding molecules of this invention consist of the multivalent binding molecules of Table 1. In an embodiment of this invention the multivalent binding molecule comprises a first polypeptide comprising SEQ ID
NO. 77 and a second peptide comprising SEQ ID NO: 79. In an embodiment of this invention the multivalent binding molecule comprises a first polypeptide comprising SEQ ID
NO: 81, or and a second peptide comprising 83. In an embodiment of this invention the multivalent binding molecule consists essentially of a first peptide comprising SEQ ID NO:
77 and a second peptide comprising SEQ ID NO: 79 and binds to FZD2 and LRP 5/6. In an embodiment of this invention the multivalent binding molecule consists essentially of a first peptide comprising SEQ ID NO: 81 and a second peptide comprising SEQ ID NO: 83 and binds to FZD7 and LRP 5/6. In an embodiment of this invention the multivalent binding molecule consists of a first polypeptide consisting of SEQ NO: 77 and a second polypeptide consisting of SEQ ID NO: 79. In an embodiment of this invention the multivalent binding molecule consists of a first polypeptide consisting of SEQ
ID NO: 81 and a second polypeptide consisting of SEQ ID NO:83.
[0069] In an embodiment of this invention the multivalent binding domains comprise one or more of the VL and VH domains of the molecules of Table 1. In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist essentially of one or more the VL and VH domains of the molecules of Table 1. In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist of one or more of the VL and VH domains of the molecules of Table 1. In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise VH
and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to VH
and VL of the molecules set forth in Table 1 and retain binding to the antigen bound by the molecules set forth in Table 1. In an embodiment of this invention the multivalent binding domains comprise one or more of the VL and VH domains of SEQ ID NOS: 77 and 79 that bind FZD2. In an embodiment of this invention the multivalent binding domains comprise one or more of the VL and VH domains of SEQ ID NOS: 81 and 83 bind FZD7.
100701 In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist essentially of one or more of the VL and VH
domains of SEQ
ID NOS:77 and 79 that bind FZD2. In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist essentially of one or more of the VL
and VH domains of SEQ ID NOS:81 and 83 that binds FZD7.
100711 In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist of one or more of the VL and VH domains of SEQ
ID NO: 77 and 79 that binds FZD2. In an embodiment of this invention the multivalent binding domains of the multivalent molecules consist of one or more of the VL and VH domains of SEQ ID
NO: 81 and 83 that binds FZD7.
100721 In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise VH and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to VH and VL domains of SEQ ID NOS: 77 and 79 and retain binding of FZD2. In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise VH and VL domains that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to VH and VL domains of SEQ ID NOS:

and 83, and retain binding to FZD7.
100731 In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise one or more complementarity determining regions (CDRs) of the molecules set forth in Table 1. In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to CDRs of the molecules set forth in Table 1 and retain binding to the antigen bound by the molecules set forth in Table 1.
In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise one or more complementarity determining regions (CDRs) of SEQ
ID NO:
77, 79, 81, or 83. In an embodiment of this invention the binding domains of the multivalent molecules described herein comprise CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98%
or 99% identical to CDRs of SEQ ID NO: 77 or 79 and retain binding to FZD2 or comprise CDRs that are at least 75%, 80%, 85%, 90%, 95%, 98% 01 99% identical to CDRs of SEQ
ID NO: 81, or 83 and retain binding to FZD7.
[0074] The FZD receptor bound by the multivalent binding molecules of this invention may be FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10, The FZD

receptor may be FZD1, FZD2, FZD4, FZD5, FZD7 or FZD8. The multivalent binding molecules may bind to only one FZD receptor or may be pan-specific binding to more than one FZD receptor. The FZD multivalent binding domain may bind to e.g., FZD1, FZD2, FZD4, FZD5, FZD7 and FZD8. The FZD multivalent binding domain may specifically bind to one FZD receptor, e.g., FZD2, FZD4, FZD5, or FZD6.
100751 In an embodiment of this invention the FZD binding domain is monospecific and binds to a single epitope on a FZD receptor. In an embodiment of this invention the FZD
binding domain is bispecific and binds to two epitopes on an FZD receptor.
[0076] The co-receptor binding domain may bind to any Wnt co-receptor, e.g., LRP5/6, or ROR1/2. The multivalent co-receptor binding domain may bind to, e.g., LRP5/6, PTK7, ROR1/2, RYK, GPR12, TSPAN12 or CD133. In an embodiment of this invention the co-receptor multivalent binding domain binds to LRP5 or LRP6.
100771 In an embodiment of this invention the co-receptor multivalent binding domain binds to a single epitope on a co-receptor, e.g., an epitope of LRP5/6 that binds Wntl or Wnt3. In an embodiment of this invention the co-receptor multivalent binding domain binds to two epitopes within a co-receptor, e.g., an epitope on LRP5/6 that binds to Wntl and an epitope that binds to Wnt3 The Wnt co-receptor bound by the multivalent binding molecules of this invention may be LRP5 or LRP6, PTK7, ROR1, ROR2, RYK, GPR124, TSPAN12 or CD133.
100781 In an embodiment of this invention the multivalent binding molecule comprises a Fc domain, wherein the Fc domain is the Fc domain of an immunoglobulin or a fragment thereof comprising the CH3 domain. In an embodiment of the invention the immunoglobulin is an IgG. In an embodiment of this invention the IgG is an IgGi.
100791 An embodiment of this invention is a method for activating a Wnt signaling pathway in a cell, comprising contacting a cell having a FZD receptor and a Wnt co-receptor with a multivalent binding molecule of this invention in an amount effective to activate Wnt signaling 100801 In an embodiment of this invention at least one of the multivalent binding domains comprises an scFv that binds the FZD receptor or co-receptor, or comprises a ligand of the FZD receptor or co-receptor or a fragment of said ligand. In an embodiment of this invention at least one of the multivalent binding domains does not comprise an scFv that binds the FZD receptor or co-receptor and does not comprise a ligand of the FZD receptor or co-receptor or a fragment of said ligand.
100811 In an embodiment of this invention the FZD
multivalent binding domains comprise a FZD diabody and the co-receptor multivalent binding domain comprises a co-receptor diabody wherein the diabodies comprises two peptides each comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the binding domain is formed by pairing of the VH and the VL from one peptide to the VL
and VH of the other peptide thereby forming the binding domains.
100821 The VH and VL of the FZD binding domain may be derived from an antibody that binds the FZD receptor and antagonizes Writ signaling or inhibits binding of a Wnt ligand to the FZD receptor. The VII and VL of the FZD binding domain may be derived from an antibody that binds the FZD receptor without antagonizing or inhibiting binding of a Wnt ligand to the FZD receptor.
100831 The VH and VL of the co-receptor binding domain may be derived from an antibody that binds the co-receptor and antagonizes Wnt signaling or inhibits binding of a Wnt ligand to the co-receptor. The VH and VL of the co-receptor binding domain may be derived from an antibody that binds the co-receptor without antagonizing Wnt signaling or inhibiting binding of a Wnt ligand to the co-receptor.
100841 In the multivalent binding molecules of this invention one or both of the binding domains may be bivalent and one or both of the bivalent binding domains may be bispecific for the FZD receptor or for the co-receptor. In an embodiment of this invention both binding domains are bivalent and bispecific, each binding domain binding to two different epitopes on their respective target FZD receptor or co-receptor. For example, the binding molecule may comprise a FZD binding domain that is bivalent and bispecific (binding to two different epitopes) for FZD receptors, or the binding molecule may comprise a co-receptor binding domain that is bivalent and bispecific for a co-receptor.
100851 In an embodiment of this invention the FZD binding domain is attached to the N-terminus of the Fc domain of the multivalent binding molecule and the co-receptor binding domain is attached to the C-terminus of the Fc domain. In an embodiment of this invention the FZD binding domain is attached to the C-terminus of the Fc domain of the multivalent binding molecule and the co-receptor binding domain is attached to the N-terminus of the Fc domain.
100861 Also an embodiment of this invention are the nucleic acid molecules encoding the multivalent biding molecules described herein, e.g. the multivalent binding molecules of Table 1, e.g. SEQ D NO, 76 and SEQ ID NO: 78, or SEQ ID NO: 80 and SEQ ID NO:
82, their VH and VL domains (e.g., SEQ ID NO: 84, 85, 86 and 87), and diabodies comprising the VL and VII domains, including expression cassettes and vectors comprising the nucleic acid molecules that encode the multivalent binding molecules, their VH, and Fc domains, and diabodies comprising such VL and VH. The nucleic acid molecules can be inserted into a vector and expressed in an appropriate host cell and then the multivalent binding molecules isolated from the cells using methods well known in the art. As used in this invention, the term "vector" refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, e.g., a nucleic acid sequence encoding the multivalent binding molecules described herein. The vector that can express protein when inserted with a polynucleotide is called an expression vector. Vectors can be inserted into the host cell by transformation, transduction, or transfection, so that the carried genetic substances can be expressed in the host cell. Vectors are well known to the technical personnel in the field, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1 derived artificial chromosome (PAC); phage such as Xphage or M13 phage and animal viruses etc.
Animal viruses may include but not limited to, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e. g. herpes simplex virus), chicken pox virus, baculovirus, papilloma virus, and papova virus (such as SV40). A vector can contain multiple components that control expression of the multivalent binding molecules described herein, including but not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., a CMV promoter, signal peptides, e.g., TRYP2 signal peptide, transcription initiation factor, enhancer, selection element, and reporter gene. In addition, the vector may also contain replication initiation site(s).
[0087] As used in this invention, the term "host cell"
refers to cells that can import vectors, including but not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 drosophila cells and S1E9, or animal cells, including human cells, e.g., fibroblast cells, CHO
cells, COS cells, NSO
cells, HeLa cells, BHK cells, or HE1C293 cells.
[0088] An embodiment of this invention is a pharmaceutical composition comprising a FZD agonist described herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise an additional agent that activates a Wnt pathway, e.g., a Nonin or R-Spondin. The pharmaceutical composition may consist or consist essentially of the multivalent binding molecules described herein and a pharmaceutically acceptable carrier or excipient. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.
R. Gennaro, Mack Publishing Company, Easton, Pa, 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the FZD agonists being administered.
[0089] Wnt-signaling is a ubiquitous pathway that modulates cellular and tissue differentiation. For example, in regards to eye development a particular Wnt-pathway, the Norrin-FZD4 pathway, has been identified as playing a role in retinal angiogenesis. Signaling through Norrin-FZD4 pathway is necessary for development and maintenance of retinal vasculature. Mutations affecting genes of this pathway may result in several pediatric vitreoretinopathies, such as Norrie Disease, Familial Exudative Vitreoretinopathy (FEVR), and Pseudoglioma and Osteoporosis Syndrome. Additionally, Retinopathy of Prematurity (ROP) has been associated with mutations in this pathway, and Wnt-pathway mutations have been reported in Coats Disease and Persistent Fetal Vasculature (PFV). The Nonin-FZD
pathway is also associated with CNS blood vessel development. Genetic ablation of the Norrin, FZD4, Lrp5 and the co-receptor Tetraspanin-12 (Tspan-12) result in defective angiogenesis and bather disruption in both retinal and cerebellar vessels (Cho et at. (2017) Neuron 95, 1056-1073; Zhou et al., (2014) J Clin Invest 1241825-3846). It is specifically contemplated herein that the FZD4 agonists of this invention, particularly the FZD4 FLAgs comprising a FZD4 binding domain on one end of the Fe receptor and a binding domain for LRP5 and/or LRP6 on the other side of the Fe domain will strengthen bather function and facilitate angiogenesis, e.g., treatment with the FZD4 FLAgs will facilitate the development and maintenance of retinal vasculature and/or the blood retinal bather (BRB) and the blood brain bather (BBB). Thus an aspect of this invention is a method for promoting and/or maintaining retinal vasculature by treating eye tissue, e.g., retinal tissue, with an effective amount of a FZD4 FLAgs through local or systemic administration. Also an aspect of this invention is a method for promoting and/or maintaining BBB vasculature by treating the BBB with an effective amount of a FZD4 FLAgs following systemic administration. A
further aspect of this invention is a method for treating a subject having a disorder characterized by reduced retinal or brain angiogenesis by administering to such subject an effective amount of a FZD4 FLAgs, wherein the effective amount is an amount sufficient to increase retinal or brain angiogenesis in such subject. The subject may be a fetus.
100901 Pathologically low levels of Wnt signaling have been associated with osteoporosis, polycystic kidney disease and neurodegenerative diseases.
Controlled activation of Wnt pathway has been shown to promote regenerative processes such as tissue repair and wound-healing. Zhao J, Kim KA, and Abo A, Trends Biotechnol. 27(3):131-6 (Mar.
2009).
See also, Logan CY and Nusse It, Annu. Rev. Cell. Dev. Biol. 20:781-810 (2004); Nusse R., Cell Res. 15(1 ):28-32 (Jan. 2005); Clevers H, Cell 127(3):469-80 (3 Nov.
2006). Proof- of-concept experiments have been done to show the role of Wnt signaling in osteoporosis or mucositis. Furthermore, it has been suggested that increasing of Wnt signaling might be beneficial for the treatment of diabetes and other metabolic diseases_ Decreased Wnt signaling has been associated with metabolic disease. Loss-of-function LRP6R611c mutation results in early coronary artery disease, metabolic syndrome and osteoporosis in human. Main A et al, Science 315:1278 (2007). "LRP5 loss-of-function mutation is associated with osteoporosis, impaired glucose metabolism and hypercholesterolaemia in human."
Saarinnen et al., Clin Endocrinol 72:481 (2010). Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both LRP5 and apoE. Magoori K. et al., JBC 1 1331 (2003). LRP5 is essential for normal cholesterol metabolism and glucose-induced insulin secretion in mice. Fujino et al., PNAS 100:229 (2003). TCF7L2 variant confers risk of type 2 diabetes. Grant et al., Nat Genet 38.320 (2006); Florez et at., N Engl J Med 355:241 (2006). An increase of Wnt signaling can be beneficial for treating metabolic diseases.
Accordingly, the administration of the multivalent binding molecules of this invention to a subject with metabolic disease is useful for treating the subjects metabolic disease.
[0091] Inflammatory bowel disease (B3P) is a group of inflammatory conditions of the colon and small intestine. The major types of ITID are Crohn's disease and ulcerative colitis.
RSP01 protein has been shown to ameliorate inflammatory bowel disease in an animal model. Zhao J et al., Gastroenterology 132:1331(2007). Accordingly, the administration of the multivalent binding molecule of this invention; e.g., a multivalent binding molecule that binds to FZD7, e.g., 12735-KM- 2539-2542, to a subject with TBD is useful for treating the subject's IRD.
[0092] Thus, an embodiment of this invention is a method for treating a subject having a condition associated with reduced Wnt signaling comprising administering to a subject in need thereof an effective amount of the FZD agonists of this invention. The condition may be e.g., osteoporosis, polycystic kidney disease, neurodegenerative diseases, mucositis, short bowel syndrome, bacterial translocation in the gastrointestinal mucosa, enterotoxigenic or enteropathic infectious diarrhea, celiac disease, non-tropical sprue, lactose intolerance and other conditions where dietary exposures cause blunting of the mucosal villi and malabsorption, atrophic gastritis and diabetes, bone fracture, tissue regeneration, e.g. tissue repair and wound healing, as well as metabolic diseases such as diabetes, and melanoma, Examples of damaged tissue that can be treated using methods of the invention include, but are not limited to, intestinal tissue, cardiac tissue, liver tissue, kidney tissue, skeletal muscle, brain tissue, bone tissue, connective tissue, and skin tissue. The multivalent binding molecules of this invention can be administered to a subject with a disease or condition characterized by a low Wnt signaling. The multivalent binding molecules of the invention are administered to the subject in an amount effective to increase Wnt signaling and to ameliorate the disease or condition in the subject.
100931 Mucositis is a clinical complication of cancer therapy. Mucositis is caused by the cytotoxic effects of irradiation or chemotherapy on fast proliferating cells.
Mucositis consists of epithelial damage mainly affecting the intestinal and oral mucosa. Clinical signs are severe pain of the oral cavity, nausea, diarrhea, malnutrition, and, in severe cases, sepsis and death.
The symptoms can often lead to dose limitation of cancer therapy. There are no currently available treatments for oral or gastrointestinal- mucositis associated with chemotherapy or radiation therapy for solid tumors.
[0094] Oral mucositis is a common and often debilitating complication of cancer treatment. 50% of patients undergoing radiotherapy for head and neck cancer and 10- 15% of patients treated with 5-FU get grade 3-4 oral mucositis. RSPO1 has been shown to ameliorate oral mucositis in an animal model. Zhao J et at., PNAS 106:2331 (2010).
[0095] Short bowel syndrome (SBS) results from functional or anatomic loss of extensive segments of small intestine, so that digestive and absorptive capacities are severely compromised. Each year, many people undergo resection of long segments of small intestine for various disorders, including trauma, inflammatory bowel disease, malignancy, mesenteric ischemia and others. Various nonoperative procedures such as radiation can cause functional short-bowel syndrome. Current therapies for short-bowel syndrome include dietary approaches, total parenteral nutrition (TPN), intestinal transplantation, and nontransplantation abdominal operations. Although these treatments have contributed to the improved outcome of SBS patients, they only partially correct the underlying problem of reduced bowel function. No current therapy can accelerate the recovery of remaining small intestine in SBS
patients. See, Seetharam and Rodrigues, The Saudi Journal of Gastroenterology 17, 229-235 (201 1).
[0096] The adult mammalian gut constitutes one of the most rapidly self-renewing tissues, in which the intestinal mucosa comprises a continuous structure folded into the proliferative crypts and the differentiated villi. In response to mucosal disruption, the host initiates a healing response resulting in restoration of mucosal integrity and regeneration of the mucosal architecture. This process is heavily dependent on the proliferation of intestinal stem cells. Neal et al., Journal of Surgical Research 167, 1-8 (2010); van der Flier and Clevers, Annual Review of Physiology 71 , 241-261 (2009).
[0097] Therefore, the factors that regulate the activity of intestinal stem cells play a dominant role in the ability of the host to respond to injury within the intestinal tract. Because Wnt proteins are the most important growth factors that support the proliferation of intestinal stem cells, enhancing Wnt signaling will increase the proliferation of intestinal epithelium.
This will lead to increased number of small bowel villi and increased mucosal absorptive surface area.
[0098] Thus, in one embodiment, the multivalent binding molecules of this invention are administered to a person with short bowel syndrome. In an embodiment of this invention the multivalent binding molecule of this invention binds FZD7, e.t, 12735-K41-2539-described herein. The multivalent binding molecules is administered in an amount sufficient to increase gastrointestinal mucosal absorptive surface area. The administration of the multivalent binding molecules of this invention has a successful outcome when the person with incident short bowel syndrome adapts to enteral feeding, or when the person with prevalent SBS absorbs nutrients from enteral feeds, or when the person decreases the amount of total parenteral nutrition required daily for the person to maintain weight.
100991 Prevention of bacterial translocation. hi one embodiment, the antibody of the invention is administered to a person at risk of septicemia caused by enteric bacteria.
The multivalent binding molecules is administered in an amount sufficient to increase gastrointestinal mucosal integrity, thus preventing enteric bacteria from passing into the bloodstream of the person. Decreased gastrointestinal mucosal integrity (as compared with the gastrointestinal mucosal integrity that is normal for the human population) is a major source of bloodstream infections and sepsis in critically ill patients. The administration of the multivalent binding molecules has a successful outcome when fewer cases of bacteremia and sepsis are observed in intensive care unit (ICU) patients than in patients to whom the multivalent binding molecules of this invention is not administered.
01001 Accelerated recovery during or after enterotoxigenic or enteropathic infectious diarrhea. Infectious diarrhea is a major pediatric problem. In one embodiment, the multivalent binding molecules of the invention is administered in an amount sufficient to shorten the time to the end of diarrhea or the time to normal bowel movements.
The multivalent binding molecules of this invention can be administered in addition to the standard of care, which includes oral or parenteral rehydration and sometimes, antibiotics.
The administration of the multivalent binding molecules has a successful outcome when decrease hospitalizations, shorten hospitalizations, or a decrease the incidence of complications of dehydration and electrolyte abnormalities are observed in pediatric patients as compared with pediatric patients to whom the multivalent binding molecules of the invention is not administered.
101011 Celiac disease, non-tropical sprue, lactose intolerance and other conditions where dietary exposures cause blunting of the mucosal villi and malabsorption. In one embodiment, the multivalent binding molecules of this invention is administered in an amount sufficient to increase mucosal absorptive surface area. The multivalent binding molecules of this invention can be administered in addition to the standard of care, which is primarily avoiding the offending foods and sometimes, dietary supplements. The administration of the multivalent binding molecules of the invention has a successful outcome when the person with celiac disease, non-tropical sprue, lactose intolerance or other condition adapts to enteral feeding, or when the person with any of the conditions absorbs nutrients from enteral feeds, or when the person decreases the amount of total parenteral nutrition required daily for the person to maintain weight.
[0102] Atrophic gastritis, specifically the form termed environmental metaplastic atrophic gastritis. Atrophic gastritis is a common condition in the elderly, currently treated with vitamin B12 injections. The patients have an increased risk of carcinoid tumors and adenocarcinoma. The administration of the multivalent binding molecules has a successful outcome when decreased the tumor incidence, in the case of carcinoid by decreasing gastrin production from the metaplastic G cells, is observed by a medical expert. The multivalent binding molecules should not be administered to the subject if a medical expert determined that if the tumors are activated by increases in the Wnt pathway.
[0103] The FZD agonists of the present invention may be administered, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, etc.), topically, or orally.
Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. The multivalent binding molecules described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The multivalent binding molecules can also be administered together with immune stimulating substances, such as cytokines.
[0104] An embodiment of this invention includes a method for producing induced pluripotent stem (iPS) cells comprising culturing a somatic cell under conditions suitable for reprogramming the somatic cell wherein said culturing conditions further comprise a multivalent binding molecule described herein. Method for generating pluripotent stem cells are well known in the art, see e.g., Takahashi and Yamanaka, (2006), Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126, 663-676; Takahashi et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Cell 131, 861-872; Yu et al. (2007).
Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920; US
patent no. 8,546,140, and; US patent No. 8,268,620. In an embodiment of this invention the multivalent binding molecules of this invention are included in the culture media in an amount sufficient to accelerate the generation of iPS cells.
101051 An embodiment of this invention includes a method for directed differentiation of multipotent or pluripotent stem cells (PSC) or induced pluripotent stem (iPS) cells comprising culturing the cells under conditions suitable for directed differentiation wherein said culturing conditions further comprise an effective amount of a multivalent binding molecule described herein. Studies in mouse and human PSCs have identified specific approaches to the addition of growth factors, including Wnt, which can induce PSC
differentiation into different lineages Methods for directed differentiation of PSCs comprising the activation of Wnt signaling are known in the art see e.g. Lain et al. (2014) Semin Nephol 34(4); 445-461; Yucer et at. (September 6, 2017) Scientific Reports 7, Article number 10741. It is contemplated that the multivalent binding molecules described herein can be used to effect activation of Wnt signaling pathways to direct differentiation of the PSCs.
101061 An embodiment of this invention is a method for enhancing tissue regeneration in a subject in need thereof by activating Wnt signaling in such subject by administering to the subject in need thereof an effective amount of a multivalent binding peptide described herein.
101071 An embodiment of this invention includes a method for enhancing bone healing and/or regeneration in a subject in need thereof, e.g., a subject with osteoporosis or fracture, by administering an effective amount of a multivalent binding molecule described herein. In a particular embodiment the multivalent biding molecule of this invention comprises a binding domain that binds to FZD2 and a binding domain that binds to LRP5 or/and LRP6.
The binding domains may be monovalent or multivalent, e.g., bivalent, trivalent or tetravalent, and monospecific or multispecific, e.g., bispecific. In an embodiment of this invention the multivalent binding molecules for enhancing bone healing and/or regeneration in a subject in need thereof comprise, e.g., 2890-knob-2539-2542 (SEQ ID NO:
77) and 2890-hole-2539-2542 (SEQ ID NO: 79) (together forming 2890-KJH- 2539:2542 or 2890Ag).
101081 A subject may be any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, horses, cows, dogs, cats, rodents, and the like.
Typically, the subject is human.

101091 Effective dosages and schedules for administering the multivalent binding molecules described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such FZD agonists that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of FZD agonists used and other drugs being administered. Guidance in selecting appropriate doses for FZD
agonists is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, NJ., (1985) ch.. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the inflammation in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the vector is within the skill of the art.
101101 In recent years, methods have been developed for culturing mini-organs called "organoids" that recapitulate the gross anatomy and cell type composition of different tissues.
Remarkably, full organoids can be generated from a single tissue stem cell as first demonstrated with intestinal LGR5+ stem cells isolated from a mouse. It is known that components within the media that activate the Wnt-13catenin pathway are required for organoid derivatization, growth, survival and maintenance. Therefore, R-spondin and Wnt ligands, purified or provided as conditioned media are universally required to grow organoids from different tissues. However, purified Wnt proteins have generally low specific activity and are not able to sustain growth of organoids. As such those of skill in the art rely on the addition of Wnt3A conditioned media, or to the addition of small molecules, e.g., GSK3 inhibitors, to generate organoids. But the production of Wnt3A conditioned media is labor intensive, the characteristics of the conditioned media are inconsistent, and small molecule GSK3 inhibitors may robustly activate the pathway to levels that are toxic The multivalent binding molecules described herein solve these problems as they are easy to produce and purify, have consistent reproducible characteristics, and activate Wnt specifically by selectively engaging the desired FZD receptor(s) and co-receptor(s) combination.
101111 An embodiment of this invention includes a method for generating tissue organoids comprising culturing tissue in an effective amount of a multivalent binding molecule described herein. An organoid is a 3D multicellular in vitro tissue construct that mimics its corresponding in vivo organ, such that it can be used to study aspects of that organ in the tissue culture dish. Methods for generating organoids are well known in the art and epithelial organoids derived from adult stem cells in the various organs of the gastrointestinal tract, for example, almost all need agonists of Wnt signaling (among other signaling factors, including embedding in Matrigel) to both maintain the cells and to generate an in vivo¨like complement of cell types. Wnt signaling also enhances inner ear organoid development in 3D
culture, and has been used in the generation of kidney organoids, see e g , Natalie de Souza (2018) Nature Methods 15(1): 23; DeJonge et al. (2016) PLosOne 11(9), e0162508;
Akkerrnan and Defize, (2017) Bioessays 39,4, 1600244. The multivalent binding molecules of this invention can be included in the culture media of organoids in an amount sufficient to enhance their growth, survival and maintenance in culture. As such, an embodiment of this invention includes a method for enhancing the culture of tissue organoids comprising a culture medium comprising an effective amount of a multivalent binding molecule described herein.
101121 Also an aspect of this invention is a method for making the multivalent binding molecules described herein. In an embodiment of this invention the multivalent binding molecule is generated by, a) selecting an Fc domain having a C-terminus and an N-terminus b) identifying a peptide that binds to one or more FZD receptors, or identifying an antibody that binds to one or more FZD receptors, and c) identifying a peptide that binds to one or more Wnt co-receptors or identifying an antibody that binds to one or more Wnt co-receptors, d) generating a nucleic acid molecule comprising a nucleotide sequence that encodes (i) the Fc domain of step a, (ii) a nucleotide sequence that encodes the peptide of step b, or a nucleotide sequence that encodes a VL and/or a VH of the antibody of step b, or a nucleotide sequence that encodes a VL and/or a VH derived from the antibody of step b, that binds the one or more FZD
receptors, and (iii) a nucleotide sequence that encodes the peptide of step c, or a nucleotide sequence that encodes the VL and/or the VH of the antibody of step c, or a nucleotide sequence that encodes the VL and/or the VH derived from the antibody of step c, that binds to the one or more Writ co-receptors, e) expressing the nucleic acid molecule of (d) to produce a polypeptide wherein the polypeptide dimerizes to form a tetravalent binding molecule comprising (i) an Fc domain, (ii) a FZD binding domain and (iii) a Wnt co-receptor binding domain wherein, such that the FZD binding domain comprises of the peptide of step b, or the VL and/or VH of step b, and is linked to one terminus of the Fc domain, and the Wnt co-receptor binding domain comprises the peptide of step c or the VL and/or VH of step c and is linked to the other terminus of the Fc domain thereby forming the multi-specific binding molecule.
10113] The peptide that binds to one or more of the FZD
receptor may be a synthetic polypeptide, e.g., a synthetic peptide, an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin or a peptide of a naturally occurring protein that binds the FZD receptor. The naturally occurring protein may be, e.g., a Wnt, e.g., Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt-8a, Wm-813, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, Wnt-16b. The peptide of step b may be multivalent, binding to more than one site on the FZD, e.g., bivalent, trivalent of tetravalent, and may be monospecific, binding to a single epitope, or multispecific, binding to more than one epitope on the FZD.
10114] The peptide that binds to one or more of the Wnt co receptor may be a synthetic peptide, e.g., an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin, or a peptide of a naturally occurring protein that binds the Wnt co-receptor.
The naturally occurring protein may be for example, a Wnt, e.g., Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a/b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11 or Wnt-16b, or Dickkopf-1.
101151 The peptide of step c may be multivalent binding to more than one epitope on the Wnt co-receptor, e.g., bivalent, trivalent of tetravalent, and may be monospecific binding to a single epitope or multi specific binding to more than one epitope on the Wnt co-receptor.
101161 The naturally occurring protein that binds the FZD
receptor and the naturally occurring protein that binds the Wnt co-receptor may be the same protein.

[0117] In an embodiment the peptide or antibody of step b may bind FZD2 and the peptide of step c may be a peptide of Wnt5a and the antibody of step c may be an antibody that binds to a site on the co-receptor that binds to Wnt5a.
[0118] In an embodiment the peptide or antibody of step b may bind FZD4 and the peptide of step c may be a peptide of one of more of Norrin, Wntl, Wnt8, or Wnt5a and the antibody of step c may be antibody that binds to a site on the co-receptor that binds to Norrin, Wntl, Wnt8, or Wnt5a.
[0119] In an embodiment the peptide or antibody of step b may bind FZD5 and the peptide of step c may be a peptide of one or more of Wnt7a, Wnt5a, Wntl Ob, or Wnt2 and the antibody of step c may be an antibody that binds to a site on the co-receptor which site binds to one or more of Wnt7a, Wnt5a, WntlOb, or Wnt2.
[0120] In an embodiment the peptide or antibody of step c binds LRP6 and/or LRP5, e.g., the peptide may be a peptide of Norrin, Wntl and/or Wnt3a, and the antibody of step c may be an antibody that binds to a site on LRP6/LRP5 which site binds to Norrin, Wntl and/or Wnt3a.
[0121] In an embodiment the peptide or antibody of step c may bind LRP6, e.g., the peptide may be a peptide of Wnt I or Wnt3a, or both, and the antibody may be an antibody that binds a site on LRP6 that binds Wntl or Wnt3a.
[0122] In an embodiment the peptide or antibody of step c binds RORI and/or ROR2 [0123] In an embodiment the peptide or antibody of step c may bind RYK.
[0124] In an embodiment the peptide or antibody of step c may bind PTK7.
[0125] In an embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to one or more FZD receptors and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. In an embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to one or more FZD receptors without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. In an embodiment, the peptide or antibody in step (c) may be a peptide or antibody that binds to one or more of the Wnt co-receptors and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor. In an embodiment, the peptide or antibody of step (c) may be a peptide or antibody that binds to the Wnt co-receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the co-receptor. The binding domains may be linked to the Fc domain via a linker. The modular aspects of this invention allows for mixing and matching of peptide or antibody VH and VL

that bind to any given FZD receptor and Wnt co-receptor on the opposite termini of the Fc domain to generate a multivalent binding molecule that can engage multiple Frizzled receptor ¨ co-receptor complexes or to selectively engage a single Frizzled receptor-co-receptor complex to activate Wnt signaling.
101261 An embodiment of this invention is a method of making a multivalent binding molecule that activates a Wnt signaling pathway comprising a) selecting an Fc domain having a C-terminus and an N-terminus, e.g. an Fc domain of an immunoglobulin comprising a CH3 domain, e.g., an IgG, e.g., an IgGl, b) identifying an antibody having a binding specificity for one or more FZD
receptor and c) identifying an antibody having a binding specificity for a Wnt co-receptor;
d) generating a nucleic acid molecule comprising (i) a nucleotide sequence that encodes the selected Fc domain, (ii) a nucleotide sequence That encodes a VL and/or a VH derived from the antibody of step b, and (iii) a nucleotide sequence that encodes a VL and/or a VI-1 derived from the antibody of step c, d) expressing the nucleic acid molecule of (d) to produce a polypeptide which dimerizes via the Fc domain to form a multivalent binding molecule comprising (i) the Fc domain, (ii) a FZD binding domain and (iii) a Wnt co-receptor binding domain, such that the FZD binding domain is linked to one terminus of the Fc domain and the Wnt co-receptor binding domain is linked to the other terminus of the Fc domain thereby forming a multivalent binding molecule In a preferred embodiment the multivalent binding molecule is a dimer of two polypeptides encoded by the nucleic acid molecule wherein the Fc domain is in a knob in hole configuration.. One or both of the binding domains may be multivalent binding domains. The antibody of step b may be an antibody fragment that binds the FZD
receptor.
The VH and/or VL in step d)(ii) may be identical to the VH and/or VL of the antibody of step b). The antibody of step c may be an antibody fragment that binds the Wnt co-receptor. The VH and/or VL in step d)(iii) may be identical to the VH and/or VL of the antibody of step c).
101271 The multivalent molecules of this invention may be generated by dimerizing two polypeptides in a "knob-in-hole" configuration. The knob-in-hole configuration increases the modularity of this invention by facilitating the association of peptides that comprise binding moieties that bind different epitopes on a FZD receptor or co-receptor or to different members of the same FZD receptor or co-receptor family, see e.g., Figure 3A.
Methods for engineering Fc molecules via the knobs into holes design are well known in the art, see e.g., W02018/026942, inventors Van Dyk et at., Carter P. (2001) J. Immunol. Methods 248, 7-15 , Ridgway et at. (1996) Protein Eng. 9, 617-621; Merchant A. M., et al..
(1998) Nat.
Biotechnol. 16, 677-681 and; et at., (1997) J. Mol. Biol. 270, 26-35.
[0128] Another embodiment of this invention is a method for facilitating the interaction of a FZD receptor and a co-receptor on a cell thereby activating a Wnt signaling pathway in the cell comprising, a) selecting an Fc domain, or fragment thereof comprising a CH3 domain, having a C-terminus and an N-terminus b) linking a first multivalent binding domain, which binds the FZD receptor, on one terminus of the Fc domain and linking a second binding domain, which binds to the Wnt co-receptor, on the other terminus of the Fc domain thereby forming a binding molecule; c) contacting said multivalent binding molecule with the cell expressing said FZD receptor and Wnt co-receptor under conditions wherein the FZD receptor and co-receptor both bind to the multivalent binding molecule thereby activating the Wnt signaling pathway. One or both of the binding domains may be monovalent or multivalent, e.g., bivalent, trivalent, or tetravalent. The FZD
binding domain may comprise a peptide of a naturally occurring protein that binds FZD, a synthetic peptide, e.g., a affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer, or an anticalin, that binds FZD, VH and/or VL fragments that bind FZD, a scFV that binds FZD, or a diabody that binds FZD. The Wnt co-receptor binding domain may comprise a peptide of a naturally occurring protein that binds the Wnt co-receptor, a synthetic peptide, e.g., an affibody, an ankyrin repeat protein, a fibronectin repeat protein, a fynomer or an anticalin, that binds to the Wnt co-receptor, VH and/or VL fragments that bind the Wnt co-receptor, a scFV that binds the Wnt co-receptor, or a diabody that binds the Wnt co-receptor.
[0129] An embodiment of this invention is a molecule comprising an Fc domain and two binding domains, the first domain binds to a FZD receptor and the second domain binds to a Wnt co-receptor, and these two moieties are linked together by a Fc domain, or fragment thereof comprising the CH3 domain, wherein one domain is linked to the N-terminus of the Fc receptor, and the other domain is linked to the C-terminus of the Fc receptor. The binding domains may be linked to the Fc receptor either directly or via a peptide linker, e.g. a polypeptide linker, or a non-peptidic linker. Suitable linkers are well known in the art, e.g., an XTEN linker (see W02013120683, inventors Schellenberger et al.) [0130] An embodiment of this invention is a method for activating a Wnt signaling pathway comprising contacting a cell expressing a FZD receptor and its co-receptor with an effective amount of the multivalent molecules of this invention. Without wishing to be bound by theory, it is contemplated that the multivalent molecules described herein bind both the FZD receptor and its co-receptor thereby forming a complex that mimics the binding of a Wnt molecule to the FZD receptor and co-receptor(s), which in turn activates Wnt signaling pathways.
[0131] The multivalent binding molecules of this invention may be made recombinantly, e.g., by Gibson assembly (see Gibson et al. (2009).. Nature Methods. 6 (5):
343-345 and Gibson DG. (2011). . Methods in Enzymology. 498: 349-361), or the molecules may be made synthetically e.g., using a commercial synthetic apparatuses, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface.
101321 In some embodiments, the binding domains are attached to the Fc domain via a peptide linker, e.g., an XTEN linker. In some embodiments, the peptide linker comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acids. In some embodiments, the peptide linker is between 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids in length The Fc domain with or without the linker are of a length and flexibility that allows for the multivalent binding molecule to bind both the FZD receptor and its co-receptor thereby activating a Wnt signaling pathway. In an embodiment of this invention the Fc domain, or fragment thereof comprising the domain, with or without the linker is greater than 100 amino acids, greater than 125 amino acids greater than 150 amino acids, greater than 175 amino acids or greater than 200 amino acids.
101331 It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g.
polypeptides, known to those skilled in the art, and so forth.
101341 An "affinity matured" antibody or "maturation of an antibody" refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent or source antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen or to other desired properties of the molecule.
101351 By "comprising" it is meant that the recited elements are required in the composition/method/kit, but other elements may be included to form the composition/method/kit etc. within the scope of the claim. For example, a composition comprising multivalent binding molecules is a composition that may comprise other elements in addition to multivalent binding molecules, e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to the multivalent binding molecules, agents that promote the stability of the multivalent binding molecule composition, agents that promote the solubility of the multivalent binding molecule composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos.
101361 By "consisting essentially of', it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, a multivalent binding molecule "consisting essentially of' a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.
101371 By "consisting of', it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, a multivalent binding molecule "consisting of' a disclosed sequence consists only of the disclosed amino acid sequence.
101381 Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention.
The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
101391 The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function In general, antibody molecules obtained from humans relate to any of the classes IgG, 1gM., IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
101401 Three highly divergent stretches within each of the heavy chain variable domain, VH, and light chain variable domain, VL, referred to as complementarity determining regions (CDRs), are interposed between more conserved flanking stretches known as "framework regions", or "FRs". Thus, the term "FR" refers to amino acid sequences which are naturally found between, and adjacent to, CDRs in immunoglobulins. A VH domain typically has four FRs, referred to herein as VII framework region 1 (FR1), VII framework region 2 (FR2), VH
framework region 3 (FR3), and VII framework region 4 (FR4). Similarly, a VL
domain typically has four FRs, referred to herein as VL framework region 1 (FR1), VL
framework region 2 (FR2), VL framework region 3 (FR3), and VL framework region 4 (FR4).
In an antibody molecule, the three CDRs of a VL domain (CDR-L1, CDR-L2 and CDR-L3) and the three CDRs of a VH domain (CDR-H1, CDR-H2 and CDR-H3) are disposed relative to each other in three dimensional space to form an antigen-binding site within the antibody variable region. The surface of the antigen-binding site is complementary to a three-dimensional surface of a bound antigen The amino acid sequences of VL and VH
domains may be numbered, and CDRs and FRs therein identified/defined, according to the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) or the INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (LMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27.55-77). One of ordinary skill in the art would possess the knowledge for numbering amino acid residues of a VL domain and of a VII domain, and identifying CDRs and FRs therein, according to a routinely employed numbering system such as the IMGT numbering system, the Kabat numbering system, and the like.
[0141] The term "antigen-binding portion" or" antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CH1 domains; (ii) an F(a13)2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of a VH domain; and (vi) an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single contiguous chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion"
of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed (see e.g., Holliger et al. (1993) PNAS. USA 90:6444-6448).
[0142] "Affibodies" are small, single domain proteins engineered to bind to a large number of target proteins or peptides with high affinity, imitating monoclonal antibodies.
They are composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of staphylococcal protein A. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants.
See, e.g., U.S. Pat. No. 5,831,012 and Lofblom et al. FEBS Letters 584 (2010) 2670-2680.

Affibody molecules mimic antibodies have a molecular weight of about 6 kDa.
[0143] "Diabodies" as used herein are dimeric antibody fragments. In each polypeptide of the diabody, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fly fragments, the linker between the VL and VH
is too short for intramolecular pairing and as such each antigen-binding site is formed by pairing of the VH
and VL of one polypeptide with the VH and VL of the other polypeptide, see e.g. Figure 3A.
Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific. (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6,118;
Poljak, R. J., et al.
(1994) Structure 2:1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).
[0144] As used herein an "effective amount" of an agent, e.g., the multivalent binding molecules or a pharmaceutical composition comprising the molecules, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
[0145] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof, or a T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is <10pM; e.g., <100 nM, preferably <10 nM and more preferably <1 nM.
[0146] The constant region of immunoglobulin molecules is also called the fragment crystallizable region, the "Pc region" or "Fc domain." The Fe domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains and the Pc domains of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fe fragment is essential for Fe receptor-mediated activity. In an embodiment of the invention the Fe domain of the multivalent molecule is engineered such that it does not target the cell binding the multivalent molecule for ADCC or CDC-dependent death. In an embodiment of the invention the Fe domain of the multivalent binding molecule is a peptide dimer in a knob-in-hole configuration. The peptide dimer may be a heterodimer.
[0147] The terms "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
[0148] "LRP", "LRP proteins" and "LRP receptors" is used herein to refer to members of the low density lipoprotein receptor-related protein family. These receptors are single-pass transmembrane proteins that bind and internalize ligands in the process of receptor-mediated endocytosis. LRP proteins LRP5 (GenBank Accession No. NM 002335.2) and LRP6 (GenBank Accession No. NM 002336.2) are included in a Wnt receptor complex required for activation on the Wnt-acatenin signaling pathway.
[0149]
The term "polypeptide fragment"
as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full length cDNA sequence.
[0150] As used herein the term "paratope" includes the antigen binding site in the variable region of an antibody that binds to an epitope.
[0151] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest.
Such treatment is desirably performed prior to complete loss of function in the affected tissue&
The subject therapy may be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
[0152] The ability of the multivalent binding molecules of this invention to activate Wnt signaling can be confirmed by a number of assays. The multivalent binding molecules of this invention typically initiate a reaction or activity that is similar to or the same as that initiated by the FZD receptor's natural ligand. The multivalent binding molecules of this invention activates the Wnt signaling pathways, e.g., the canonical Wnt-13catenin signaling pathway. As used herein, the term "activates" refers to a measurable increase in the intracellular level of a Wnt signaling pathway, e.g., the Wnt-13catenin signaling pathway, compared with the level in the absence of a FZD agonist of the invention.
[0153] Various methods are known in the art for measuring the level of Wnt-13catenin activation. These include but are not limited to assays that measure: Wnt-13catenin target gene expression; LEF/TCF reporter gene expression (such as TopFLASH, superTopFLASH, pBAR); fIcatenin stabilization; LRP5/6 phosphorylation; Axin translocation from cytoplasm to cell membrane and binding to LRP5/6. The canonical Wnt-f1catenin signaling pathway ultimately leads to changes in gene expression through the transcription factors TCF1, TCF7L1, TCF7L2 and LEE The transcriptional response to Wnt activation has been characterized in a number of cells and tissues. As such, global transcriptional profiling by methods well known in the art can be used to assess Wnt-13catenin signaling activation.
[0154] Changes in Wnt-responsive gene expression are generally mediated by TCF and LEF transcription factor& A TCF reporter assay assesses changes in the transcription of TCF/LEF controlled genes to determine the level of Wnt-13catenin signaling. A
TCF reporter assay was first described by Korinek, V. et al., 1997. Also known as TOP/FOP
this method involves the use of three copies of the optimal TCF motif CCTTTGATC, or three copies of the mutant motif CCTTTGGCC, upstream of a minimal c-Fos promoter driving luciferase expression (pTOPFLASH and pFOPFLASH, respectively) to determine the transactivational activity of endogenous 13catenin/TCF. A higher ratio of these two reporter activities (TOP/FOP) indicates higher acatenin/TCF activity. A newer and more sensitive version of this reporter is called pBAR and contains 12 repeats of the TCF motifs (Biechele and Moon, Methods Mol Biol. 2008;468:99-110, FMB): 19099249).
[0155] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed.
(Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed.
(Ausubel et al.
eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley &
Sons 1996);
Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);
Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I.
Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998)..

[0156] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of antibody, wherein these domains are present in a single polypeptide chain.
Generally, the Fy polypeptide further comprises a polypeptide linker between the VH and VL
domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).
[0157] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art Standard techniques are used for recombinant DNA_, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients_ EXAMPLE I
1. Development of multivalent FZD agonists.
[0158] To make a multivalent binding molecule having a first binding domain comprising a FZD diabody and a second binding domain comprising a co-receptor diabody, we identified FZD specific antibodies from a synthetic Fab phage library (Library F; see US
publication no. 2016/0194394, inventors Sidhu et al.) by selecting for those that bound to the cysteine rich domain (CRD) of FZD receptors using conventional phage-display technologies.
Affinity or specificity maturation was carried out as needed. For example, a pan-FZD binding antibody, #5019 (which recognizes FZD1, 2,4, 5, 7 and 8), was maturated from a derived antibody using the FZD4 CRD as antigen. Our previous work also identified several antibodies that are completely specific for FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) or for FZD5 (2928) (see, e.g., US20160194394, inventors Sidhu et al_ and W02017127933A1, inventors Pan et al.).
101591 These FZD antibodies were used to prepare a FZD
specific diabody. A diabody is an antibody form similar to single chain variable fragment (scFv), but it is a dimer of two peptides each encoding a VL and VH, however, unlike a scFv the linker between the VH and VL within the polypeptides is too short to allow for intramolecular complementation between the VH and VL domains. Therefore, the VH-VL fragment of one polypeptide dimerizes with the VH-VL fragment of another polypeptide in such a way to functionally reconstitute two antigen binding paratopes. Diabodies were generated having paratopes that were identical or non-identical, by forming dimers of the polypeptides having the same VL and VII thereby forming homodiabodies, or forming dimers from two polypeptides having different VL and VH domain thereby forming heterodiabodies .
101601 LRP6 antibodies were also selected from a synthetic antibody library by selecting those that bound the recombinant extracellular domain (ECD) of human LRP6.
Five Fab with unique CDR regions were identified. After converting to IgG forms, they all display human LRP6 binding as well as mouse LRP6 binding. No LRP5 binding was detected via ELISA, demonstrating these antibodies are LRP6 specific (Figure 1A). LRP6 ECD
contains four (3-propeller motifs that alternate with four epidermal growth factor (EGF) like repeats. The first two (I- propeller motifs are thought to be involved in Wnt1 binding and the last two are thought to be involved in Wnt3 binding, thus creating two potential epitopes for antibody binding See Figure 6A. Epitope binding results suggest that these five antibodies bind two separate sites on LRP6 and could be divided into two groups with antibodies 2538, 2542, and 2543 binding the Wnt1 binding site and 2539 and 2540 binding the Wnt3 binding site on LRP6. In general, an antibody binding to the LRP6-Wntl site would be expected to block Wntl-induced Wnt pathway activation.
101611 To prepare the Fe N-terminal binding domain containing a homodiabody specific for a FZD, the VH and VL fragments, VH-1, VH-2, VL-1 and VL-2, of the selected FZD
antibodies were amplified by PCR from the corresponding phagemid templates and isolated.

Gibson assembly was then utilized to introduce the isolated fragments (VH-1 and VL-2) into an EcoR1/XhoI precut vector containing an Fc-knob region (pSCST backbone) (see Gibson et al. (2009). . Nature Methods. 6 (5): 343-345 and Gibson DG. (2011). Methods in Enzymology. 498: 349-361). Gibson assembly was also utilized to introduce the fragments (VH-2 and VL-1) into the EcoRIDChoI precut vector containing an Fc-hole region. Correct assembly was validated using DNA sequencing. The two plasmids (one pair, Fc-knob and Fc-hole) were then used to introduce the second binding domain at the C-terminus of the Fc domain.
[0162] The Fc-knob and Fc-hole configuration was needed to generate multivalent binding domains wherein one of the binding domains was a heterodiabody.
However, the Fc-knob and Fc-hole configuration was not needed to prepare binding molecules that comprise homodiabodies on both the N and C termini of the Fc domain and thus for such binding molecules the VHs and VLs were linked to a wild-type Fc region and only one plasmid was used to generate the VH-VL containing polypeptides to form the homodimer.
Optionally, a linker, e.g. a peptide linker, or a non-peptidic linker, can be present between the binding domains and the Fc domain [0163] To generate the C-terminal binding domain, an LRP5/6 antibody was identified and an LRP5/6 diabody was generated following the same protocol as described above for generating the FZD diabody. The C-terminal binding domain was generated by PCR

amplifying the VH-3, VH-4, VL-3 and VL-4 fragments from the corresponding phagemid template for the LRP antibody and then isolating the amplified fragments. As described above, Gibson assembly was then utilized to introduce the VH-3 and VL-4 fragments into the PpuMI/Bamill site of the Fc-knob plasmid described above. Gibson assembly was used to insert the other VH-4 and VL-3 fragments into the PpuMI/BamHI cut of the Fc-hole plasmid [0164] Two plasmids (one pair, Fc-knob and Fc-hole) with differing VL and WI
sequences were used to generate a FZD or co-receptor binding domain that was bispecific, i.e., capable of binding to two different sites. Because a knob-into-hole configuration was not needed to generate a dimer having monospecific binding domains, only a single plasmid containing the wild-type Fc sequence was used if each of the binding domains were to be monospecific.
[0165] Figure 9A depicts a plasmid encoding the peptide comprising an Fc region comprising a "knob" mutation, the VH and VL of panFZD antibody 45019, and the VL of LRP antibody #2542 and the VH of LRP antibody #2539. Figure 9B depicts a plasmid encoding the peptide comprising a nucleic acid encoding Fc region comprising a "hole"
mutation, the VH and VL of panFZD antibody #5019, and the VH of LRP antibody #2542 and the VL of LRP antibody #2539. The peptides encoded by these plasmids form a heterodimer having a multivalent binding site comprising a homodiabody derived from the pan specific FZD antibody #5019 and a multivalent binding site comprising a bispecific heterodiabody produced by the pairing of VL of LRP antibody #2539 and VH of LRP
antibody #2542 from one peptide with the VII of LRP antibody #2539 and the VL
of LRP
antibody #2542 of the other peptide.
[0166] The resulting plasmids were then sequenced and the sequenced-verified plasmids were prepared using a PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen) according to manufacturer's instructions. The plasmids were then transfected into Expi293F cells (Thermo Fisher Scientific) and FectoPRO Reagent (Polyplus) was used for antibody expression according to the manufactory's instructions Typically, a scale of 200m1 cell was used for a small batch antibody production.
[0167] Typically, 80h after transfection, the Expi 293F
cell culture medium was harvested by centrifugation to pellet the cells and cellular debris. The supernatant was transferred to a clean bottle and buffered with 10xPBS buffer. After lh incubation with appropriate amount of Protein A beads (GE Healthcare), the beads were washed and the binding molecules were eluted according to the manufacturer instruction.
Finally, the buffer was exchanged into PBS.
2. Heterodimeric multivalent binding molecules 101681 Using the methods described above, we also generated tetravalent heterodimeric molecules which contain intact bispecific diabodies fused to each of the N-terminus and C-terminus of Fc domain (Knob/Hole), (Figure 2A and 3A). In particular, we generated a tetravalent binding molecule having a FZD binding homodiabody derived from antibody 5019 on the N-terminus of the Fc domain and a homodiabody derived from LRP6-W1 antibody 2542 (5019-Fc-2542 ) or LRP6-W3 antibody 2539 (5019-Fc-2539) on the C-terminus of the Fc domain Surprisingly, both tetravalent molecules activated the Wnt pathway, but 5019-Fc-2542 had much less efficacy (FIG. 3C). Without wishing to be bound by theory this difference may reflect differences in capacity of LRP6-W1 and binding to activate Wnt signaling. Wnt binding of the LRP6-W3 site has been observed to be more effective in activating Wnt signaling than Wnt binding to the LRP6-W1 site.
101691 We also generated a tetravalent trispecific binding molecule having a FZD

binding homodiabody derived from antibody 5019 on the N-terminus of the Fc domain and a LRP heterodiabody derived from LRP6-W1 antibody 2542 and LRP6-W3 antibody 2539 on the C-terminus of the Fc domain (5019-KM-2539-2542, named as 5019Ag) (Figure 5).
5019Ag is unexpectedly effective in activating Wnt signaling as compared to the molecules having a monospecific LRP6 homodiabody (Figure 3C). Nanomolar amounts of all three forms activate Wnt signaling determined by pBAR luciferase reporter assays (Figure 3D), indicating they are effective Wnt mimics. Without wishing to be bound by theory it is contemplated that engagement of a strong Wnt3A site and a weak Wnt1 site together is more effective than engagement of two strong Wnt3A sites. The two best multivalent binding molecules having an FZD binding domain and a LRP binding domain, "FLAgs", had single-digit nanomolar potency (EC50 ¨5 nivI), which was virtually identical to the potency of purified Wnt3A, and displayed a bell-shaped dose response profile (Figure 11D). We interpret this as indicating that maximal stimulation requires multivalent binding of the FLAg and that decreased efficacy at higher concentrations is likely attributable to monovalent binding to either FZD or LRP6. We treated RKO cells, which express low levels ofpcatenin (Major et al. Science. 316, 1043-1046 (2007)), with FP-41-L6E+3 which caused dose- and time-dependent increases in 13catenin protein levels and phosphorylation of DVL2, a hallmark of Wnt-FZD pathway activation (FIG. 11E and FIG. 11F). Thus, tetravalent FLAgs are modular, engineerable, human Ab modalities that function as synthetic agonists of FZD
and LRP6.
[0170] To confirm the engineered affinity and specificity of the optimal FLAg FP+P-L61+3, we used Bio-Layer Intetferometry (BLI) to measure its binding kinetics to nine of the 10 human FZD CRDs and to human LRP6 ECD (FIG. 12A and FIG. 12B). The FLAg bound with affinities in the picomolar range (KD = 10-800 pM) to the six FZDs recognized by the FZD diabodies derived from the parent pan-FZD paratope (Pavlovic et at. 2018) but did not bind detectably to the other three FZDs. Moreover, affinity for LRP6 was in the nanomolar range (10 = 12 nM) (FIG. 12B). We then used BLI to assess FLAg binding to various Fc receptors.
[0171] The FLAg behaved similarly to a conventional IgG
and interacted with FcRn in a dose and pH dependent manner (FIG. 12C). Natural IgGs bind to FcRn at pH 6 but not at pH

7.4, and this enables recycling during pinocytosis and consequent long half-life in vivo. The FLAg also behaved similarly to the IgG for interaction with other Fc effectors including complement (Clq), the natural killer cell marker CD16a, the B cell marker CD32a, and the monocyte and macrophage marker CD64 (FIG. 12D. We conclude that the FLAg contains a functional Fc moiety that should confer effector functions and long half-life in vivo.
101721 The modular design of the tetravalent FP P-L61 3 FLAg allowed us to dissect the contributions of each of the four paratopes to the intrinsic agonist activity by replacing each with a null paratope binding to the irrelevant antigen maltose-binding protein (MBP). We generated "mono-binding" molecules comprising an Fc domain and an FZD binding domain attached to one Fc domain terminus and LRP binding domain attached the other Fc domain terminus, but rather than having two binding sites for FZD or LRP within the diabodies, the binding domains have only a single or mono binding site, and one control maltose-binding-protein binding site, "MBP". One MBP binding site was introduced into at least one binding domain of the molecules to generate five mono binding molecules. The 5019-MBP-IQH-2539-2542, which contains one FZD and one MEP binding site in the N-terminus, still activates the Wnt pathway, but has an 8-fold decrease in efficacy as compared to 5019Ag (FIG. 3E). Similarly, the 5019-K/H-2539-MBP, which retains only one LRP6-W3 site in the C-terminus, exhibits much less Wnt activation as compared to 5019A8 (Figure 3E). Minimal agonistic activity was detected for the two MBP-FZD/MBP-LRP6 molecules 5019-MBP-K/H-2539-MBP and 5019-MBP-K/H-MBP-2542 and the molecules having one LRP6-W1 diabody, 5019-K/H-MBP-2542 (Figure 3E). The results of thesel3catenin signaling assays showed that maximal stimulation was reduced significantly by disabling one anti-FZD
paratope or the anti-LRP6 paratope for the WNT1 binding site and was completely ablated by disabling the anti-LRP6 paratope for the WNT3A binding site or by simultaneously disabling one anti-FZD paratope and either of the anti-L1IP6 paratopes. We also substituted an anti-LRP5 paratope targeting the WNT3A binding site for the anti-LRP6 paratope targeting the WNT1 binding site to generate a molecule (F'+P-L5/63) that could recruit both co-receptors and observed activity similar to that of FP+P-L61+3 (FIG. 3F, EC50 = 4 nM).
Taken together, these data showed that optimal agonist activity is achieved with a molecule capable of recruiting two FZDs through a common epitope and LRP6 through two distinct epitopes, but activity can be modulated to intermediate levels by disabling one of the anti-FZD or anti-LRP6 paratopes. Moreover, molecules that could recruit FZD and two different co-receptors were generated by combining two anti-FZD paratopes with one paratope each for LRP5 and LRP6.
101731 We also explored the requirements for geometric and spatial constraints imposed by the intermolecular diabody format by substituting diabody pairs with pairs of less constrained intramolecular single-chain variable fragments (scFvs) (FIG. 2J), Compared with FP+P-L6 1 3 a FLAg that contained anti-FZD scFvs (Fr+Ps-L6 1+3 ) exhibited similar activity, whereas activity was significantly reduced for FLAgs that contained anti-LRP6 scFvs (FP+P-L6141+3*) or scFvs at both ends (FP4I+Ps-L61.+3*). These differences in activity were not due to differences in affinity, as BLI measurements showed comparable, high-affinity binding to LRP6 and FZD isoforms regardless of whether paratopes were presented in the diabody or scFv format (FIG. 2K and FIG. 2L). Taken together, these results showed that particular stoichiometries and geometries are required for the assembly of optimal FZD/LRP6 signaling complexes, and constraints are especially precise for LRP6, which requires engagement of two distinct epitopes in a specific geometry dictated by the diabody format.
Notably, the looser constraints for FZD engagement enabled significant activation with a single anti-FZD
paratope (FIG. 2D), which opens the door for further enhancing specificity or altering signaling by recruiting a different cell surface protein through an additional paratope in conjunction with an anti-FZD paratope at the N-termini of the heterodimeric Fc.
3. Other bispecific antibody forms 101741 Bispecific molecules comprising a FZD binding domain of antibody #5019 and LRP6-W1 binding domain of antibody #2942 (5019/2942) or LRP6-W3 binding domain of antibody #2539 (5019/2539) on the same terminus of an Fe domain were constructed and the corresponding proteins were purified (Figure 2A) and assayed for activation of Wnt signaling using pBAR luciferase reporter assays. These molecules failed to activate Wnt signaling.
Notably, both bispecific molecules antagonized the activity of the Win ligand (Figure 28).
Without wishing to be bound by theory, the distance and flexibility between the two paratopes of these bispecific molecules might not recruit the FZD and LRP6 receptor in a suitable geometry for activation.
101751 Bispecific molecules comprising a FZD diabody and an LRP diabody attached to the same terminus of an Fc domain were also generated using a knob in hole configuration.
These diabodies designated 5019-2539-K/H (FZD/LRP-W3) and 5019-2542-K/H
(FZD/LRP-W1) were assayed for FZD and LRP binding and Win pathway activation. Both diabodies retained the FZD binding profile of the original antibody as well as the LRP6 binding activity (FIGS. 2D -2G). Both molecules bound individually to the FZD receptor and the LRP co-receptor. 5019-2542-K/H displayed co-binding to both FZD and LRP in solution as determined with BLI assays (FIG. 2H) but no significant co-binding was observed with 5019-2539-KM. Neither 5019-2539-KM nor 5019-2542-K/H activated Wnt signaling as determined in pBAR luciferase reporter assays, similar to the results obtained with the homo-diabodies that bound to only a FZD receptor (5019-Fc) or co-receptor (2539-Fe) (FIG.
21). Moreover, both 5019-2539-K/H (FZD/LRP-W3) and 5019-2542-K/H (FZD/LRP-W1) effectively inhibited Wnt3a mediated pathway activation (Figure 21).
4. Wnt Pathway Signaling Assay 101761 Wnt pathway activation was assayed in HEK293 cells using the pBAR luciferase reporter system that faithfully monitor the transcriptional activation of Bcatenin (Biechele and Moon, Methods Mol Biol. 2008;468:99-110, PM1D: 19099249). Briefly, HEK293T
cells stably expressing pBARLS and pSL9 Luciferase constructs were seeded in 96-well plates at 1.5E4 cells/well. 24 hours following seeding, cells were treated with the indicated FZD agonists in triplicate at indicated concentrations or PBS
vehicle control. 16.5 hours after treatment cells were lysed and luminescence was measured using Dual-Luciferase Reporter Assay System (Promega #E1960), according to manufacturer's protocol.
Firefly luminescence was normalized to Renilla luminescence for each well, to control for cell number.
101771 We assayed the agonist activity of multivalent molecules containing an N-terminal FZD diabody derived from an antibody fragment (Antibody #5019) that recognized several of the FZD receptors (FZD 1, 2, 4, 5, 7, and 8) joined via the Fc domain to an LRP binding domain on the C-terminus of the Fc domain. The C-terminal LRP binding domains comprised a diabody derived from one of two LRP6 antibodies, #2539 and #2542, which bind to the Wnt3 site and Wnt1 site respectively (FIG. 6B). Nanomolar amounts of these multivalent binding molecules, denoted 5019-Fc-2539 and 5019-Fc-2542 activated the Wnt-Bcatenin pathway (FIG. 6C), however, treatment of cells with the molecule harboring the LRP6 antibody targeting the Wnt3 site, 5019-Fc-2539, led to an approximately 10 folds higher activation when compared to 5019-Fc-2542 (200 folds vs 20 folds over background respectively) (FIG. 6C).
101781 Importantly, using a knob-hole system engineered within the Fc moiety we generated multivalent binding molecules (FIG. 1C) that contained a homodiabody for the pan-FZD binding domain on one end (#5019) and an heterodiabody forming LRP6 binding domain with binding sites for Wnt1 (#2542) and Wnt3 (#2539) 5019-K/H-2539:2542 on the other end (FIG. (SB). This configuration enabled the incorporation of 4 different binding sites within the molecule with different selectivity and affinity profiles, i.e., tetravalent and trispecific. When tested in the B-catenin luciferase reporter assay in HEK293 cells, this molecule has a 2 folds higher activation than 5019-Fc-2539 or approximately 400 folds over background (FIG. 6C).
101791 We also substituted the binding sites for LRP6 for equivalent LRP5 binding sites (diabodies derived from 2459 and 2460 antibodies which both bind LRP5) within the knob-in-hole system along with the same pan-FZD diabody that binds FZD1, 2, 4, 5, 7, 8 (5019).
This molecule 5019-K/H-2459:2460 was also able to activate the Wnt-Bcatenin pathway in HEK293T cells (FIG. 6D), albeit with lower efficacy than the agonist harboring the LRP6 diabodies.
5. Characterization of selective FZD agonists (Agonist modularity with binding domains derived from selective FZD and co- receptor antibody fragments) 101801 To assess activities of our monospecific FZD
agonists, we used cell-based assays that depend on particular FZD isforms. We prepared multivalent binding molecules that only bound to one of the ten FZD receptors. Our previous work identified several antibodies that are completely specific for FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) (see, e.g., US20160194394, inventors Sidhu et al. and W02017127933A1, inventors Pan et al.).
Multivalent binding molecules comprised a FZD binding domain that was FZD4 specific and an LRP6 binding domain comprising a bispecific heterodiabody derived from antibodies 2539 and 2542 were generated using the Fc knob-in-hole system. These molecules could activate FZD4 signaling through the 13-catenin pathway but only when co-transfected into HEK293 cells along with FZD4 cDNA. These FZD4 binding molecules could not activate FZD4 signaling or the 13-catenin pathway in non-modified HEK293T cells, which express low levels of FZD4. Thus, this experiment demonstrates the specificity of the molecules for FZD4. 5019-IQH-2539- 2542 (the pan-FZD agonist described above) can activate signaling in HEK293T cells even in the absence of FZD4 (FIG. 4A). This result is not surprising as Wnt-mediated activation of B-catenin signaling HEK293T cells occurs through FZD1, 2 and 7 (Voloshanenko et al. FASEB 2017 FASEB J. 2017 Nov; 31(11):4832-4844; PMID:
28733458) and the 5919 FZD antibody binds to all three receptors.
101811 In addition, we generated a FZD5 specific multivalent binding molecule using the binding domain of the FZD5 specific antibody 2928, which we previously characterized to bind only to FZD5 (Steinhart et al. Nat Med. 2017 Jan; 23(1):60-68, PMID:
27869803;
W02017127933A1, inventors Pan et al.). We previously demonstrated that several mutant pancreatic ductal adenocarcinoma (PDAC) cell lines are dependent only on FZD5 signaling for their proliferation (Steinhart et al. 2017, PMID: 27869803).
Indeed, genome-wide CRISPR essentiality/fitness screens in three RNF43 mutant PDAC lines showed that FZD5 was one of the most essential genes for their growth whereas PDAC cell lines with WT
RNF43 did not exhibit this requirement for FZD5. When RNF43 mutant cells are treated with a Porcupine inhibitor (PORCNi, such as LGK-974) that inhibits the palmitoylation and activity of Wnt ligands, RNF43 mutant cells stop proliferating.
101821 Co-treatment of RNF43 mutant cells with the pan-FZDag 5019-K/H-2539-2542 or with the selective FZD5 agonist 2928-K/H-2539-2542 led to robust rescue of cell proliferation blocked by LGK974. These results demonstrate that these two molecules were capable of activating FZD5 and induced Wnt signaling in these cells, thereby mimicking the action of endogenous Wnt ligands (FIG. 7B). In contrast, addition of the FZD4 specific agonist 5038-K/H-2539-2542 or a FZD2 specific agonist were unable to rescue the inhibition of proliferation mediated by LGK974.
101831 RNAseq analysis has shown that FZD2 is the predominant isoform in the mesenchymal stem cell line CH3H10T1/2 (Mouse ENCODE), suggesting that FZD2 may be responsible for the established role of Wnt proteins during osteogenic differentiation of mesenchymal cells (Day et al. Dev. Cell. 8, 739-750 (2005)). Stimulation of cells with a FZD2-specific FLAg led to robust induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with a Pan-FZD FLAg, whereas a FZD5-specific FLAg exhibited minimal activity (FIG. 7B).
6. Co-targeting with the tetravalent binding molecules 101841 In addition to mixing and matching FZD multivalent binding domains and co-receptor binding domains with an Fc domain to achieve desired combinations, the existence of tetravalent paratopes in the current system provides an opportunity for targeting two FZD
receptors and two co-receptors simultaneously with one molecule, ensuring the co-localization when applying in vivo. Considering the agonistic activity of 5019-MBP-K/H-2539:2542 shown above, the generation of a multivalent binding molecule having binding domains for selective FZD receptors by combining the binding regions within an heterodiabody at the N-terminus of the Fc domain. For example, the binding domains derived from antibody 5038 (binds FZD4) and 2928 (binds FZD5) would yield a FZD4 and FZD5 co-targeting molecule. The binding molecules can also be generated to have a co-receptor binding domain for specific or multiple co-receptors. For example, an co-targeting binding domain could be produced by combining the binding domains derived from the 2459 (binds Wntl binding site of LRP6) and 2539 (binds Wnt3a binding site of LRP6) antibodies on the C-terminal of the Fc domain. Likewise, the co-receptor binding domain may comprise a binding site for an LRP6 in combination with another co-receptor, e.g., ROR1/2, to initiate activation of both canonical and non-canonical Wnt signaling pathways in a single cell.
101851 Also contemplated herein is a multivalent binding molecule having a tissue specific binding domain derived from a tissue specific antibody which would recruit the multivalent binding molecule to a desired tissue where it would then activate Wnt signaling by binding a FZD receptor and co-receptor. This is contemplated to be particularly useful when using the multivalent binding molecules in regenerative therapeutics when desired effects may need to be restricted to a specific tissue. To summarize, the tetravalent mode allows more designing flexibility to meet versatile functional requirements.
7. Multivalent binding molecules having a FZD binding domain and co-receptor binding domain can replace Wnt ligands to sustain intestinal organoid cultures.
101861 The effect of the FZD agonists described herein on organoid survival and maintenance was assayed as follows. An 8-week old female C57BL/6 mouse was sacrificed, and small intestine crypts were harvested for organoid isolation (O'Rourke et at. 2016.
Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells. Bio Protoc. 20:4).
Organoid cultures were passed by mechanical dissociation (O'Rourke 2016) and embedded in 25111 of Growth Factor Reduced Matrigel (Coming, 356231) in a 48 well plate. Organoids were plated in triplicates for each experimental condition. Complete organoid media (O'Rourke 2016) with experimental conditions (11.1M LGK-974 +/- 40% Wnt3a conditioned media or +/- 50nM panFzd-5056 (a FZDag targeting FZD1, 2,4, 6, 7, 8 but binding to an epitope that does not compete with Wnt ligands)) was added to each well on day of passaging and changed every 2-3 days. After one week, 150 1 of Cell Titer Glo 3D
(Promega) was added to 1500 of media in each well. Organoids were lysed on a rocking platform for 30 min at RT. The luminescence reading was measured in duplicates for 20td of lysate from each well. The average luminescence reading for each condition was normalized to the DMSO
condition to calculate viability.
101871 Being pervasive stem cell niche factors, Wnts and R-spondins are required for the derivatization and maintenance of three-dimensional culture organoids from many tissues. In vitro, Wnt proteins secreted by paneth cells are sufficient to support the growth of mouse small intestine organoids in the presence of R-spondins. However, if Wnt release and activity is blocked with the PORCNi LGK974, the organoids can't proliferate and eventually die.
Herein we demonstrate that a pan-FZD multivalent binding molecule of this invention, FZDag (FP+P-L61+3) can rescue and sustain the growth of organoids in the presence of LGK974, suggesting that the molecule functionally mimics Wnt ligands (FIG. 8) and can substitute for Wnt proteins to support growth of tissue organoids,. Because Wnt ligands are integral components of the media required to grow many human tissue organoids, the antibody-derived FZD agonists of this invention are expected to promote the derivatization, survival and maintenance of organoids of different tissues when included in the culture media and thereby alleviate limitations associated with the use of conditioned media or purified Wnt proteins.

8. Multivalent binding molecules promote bone regeneration 101881 A rat closed femoral fracture model is used to evaluate the regenerative properties of multivalent binding molecules of this invention having a first multivalent binding domain that binds FZD2, and a co-receptor binding domain that binds to LRP5 or LRP 6.
The first multivalent binding domain may specifically bind FZD2, e.g., the binding domains of 2890-hole-2539-2542 and 2890-knob-2539-2542 (e.g. encoded by SEQ ID NO: 84 and 85) or may bind FZD2 and other FZD receptors.
101891 Rats are administered vehicle or the multivalent binding molecule following unilateral closed femoral mid-diaphyseal fractures (see Bonnarens, and Einhorn, J. Orthop.
Res. 2, 97-101 (1984)). Briefly, an 18-gauge syringe needle is inserted into the medullary canal through the condyles_ A transverse fracture of the femur is then created via blunt impact loading at the anterior (lateral) aspect of the thigh. One day after the fracture, rats are injected subcutaneously with either saline vehicle or multivalent binding molecules twice per week for 7 weeks. At termination, the intramedullary pins are removed and the fractured femurs will be analyzed by microCT.
101901 The multivalent binding molecules having a multivalent domain that binds FZD2, and a second multivalent binding domain that binds to LRP5 or LRP6 significantly increases regeneration of bone in this model in comparison to bone regeneration by the vehicle alone.
EXAMPLE 11-Synthetic antibodies targeting FZD and LRP6 101911 We previously applied phage display to derive hundreds of synthetic Abs using nine recombinant FZD CRDs as antigens (FZD3 CRD could not be purified) (Steinhart et al.
Nat. Med. 23 , 60(2016); Pavlovic et al. MAbs (2018), doi:
10.1080/19420862.2018.
1515565). Systematic characterization revealed a continuum of specificity profiles with some Abs displaying broad specificities, exemplified by a pan-FZD Ab (FP) that recognized FZD1/2/4/5/7/8 (FIG.11A), others displaying more restricted specificities, and some being monospecific (FIG. 11B). Functional characterization revealed that some antibodies competed with Wnt and inhibited 13catenin signaling, whereas others were non-competitive and did not interfere with Wnt signaling (FIG. 11B). In total, we fully characterized 161 anti-FZD antibodies, including 47 inhibitors of Wnt signaling. Unexpectedly as discussed herein, regardless of whether or not they competed with Wnt and inhibited Wnt signaling, all the multivalent binding molecules that we generated by using these anti-FZD
antibodies as the source of the FZD binding domains in conjunction with an LRP binding domain, e.g., a binding domain that bound to Wntl and/or Wnt3a binding sites on LRP 5/6, were agonists of the Wnt pathways.
EXAMPLE - Phenotypic effects of FLAgs in cells, organoids and animals 101921 Having established that FLAgs selectively engage FZD and LRP to activate Wnt-associated signaling pathways, we explored the phenotypic effects of these signals in progenitor stem cells (PSCs), organoids and animals_ Modulation of Wnt-f3catenin signaling activity is integral to most PSCs differentiation protocols (Huggins et al.
Methods Mol, Biol.
1481, 161-181(2016)). Treatment of human PSCs with WNT3A conditioned media, or small molecule inhibitors of GSK3, activates Pcatenin signaling, leads to primitive streak induction, and promotes mesodermal fate specification (Davidson et al. PNAS U.S.A. 109, (2012)). We evaluated FLAg activity in this context and found that treatment of human PSCs with 30 nM F"-L61+3 for three days caused robust induction of the mesoderm marker BRACHYURY and decreased expression of the pluripotency marker OCT4 to levels comparable to treatment with the GSK3 inhibitor CHIR99021 at 6 pM (FIG. 13A
and FIG.
13B).
101931 F'-L6' recognizes mouse FZDs and LRP6, and it contains an Fc that interacts with the FcRn. It is contemplated that the Fe endows the molecule with a long, Ab-like, half-life in vivo. Thus, we tested whether F""-L6'3 could interact with endogenous receptors in mice and accumulate to levels that would be sufficient to activatef3catenin signaling and mobilize endogenous stem cell activity. Within the intestinal stem cell niche, Wnt proteins secreted by mesenchymal cells induce expression of I3catenin target genes in stem cells at the bottom of the crypt to direct their self-renewal, and the target gene LGR5 is frequently used as a marker of stem cells in various tissues. Treatment of LGR5-GFP mice with ablated Wnt production and caused rapid extinction of LGR5 expression and the linked GFP
signal in crypt stem cells. Strikingly, GFP expression was rescued upon co-treatment with FP P-L61+3 by intraperitoneal injection (Figure 14 right panel. We conclude that FP+P-L61 3 has a sufficient half-life and bioavailability to enable Pcatenin activation at levels that promote self-renewal of intestinal stem cells in the absence of endogenous Wnt.
EXAMPLE IV - Materials and Methods:
1. Ab selections and screens 101941 The phage-displayed synthetic library F was used to select for Fabs that bound to Wnt receptors, as described (Persson et al. J. Mol, Biol. 425 , 803-811(2013)). Briefly, Fe-tagged ECD protein (R&D Systems) was immobilized on Maxisorp immunoplates (ThertnoFisher, catalog number 12-565-135) and used for positive binding selections with library phage pools that were first exposed to similarly immobilized Fc protein to deplete non-specific binders. After 4 rounds of binding selections, clonal phage were prepared and evaluated by phage ELISA (Birtalan et al. J. Mol. Biol. 377, 1518-1528 (2008)). Clones that displayed at least 10-fold greater signal for binding to antigen compared with Fe were considered to be specific binders that were subjected to further characterization.
2. Recombinant proteins and reagents 101951 Fe-tagged fusions of FZD1 (5988-FZ-050), FZD2 (1307-FZ-050), FZD4 (5847-FZ-050), FZD5 (1617-FZ-050), FZD7 (6178-FZ-050), FZD8 (6129-FZ-050), FZD9 (9175-FZ-050), FZD10 (3459-FZ-050) were purchased from R&D Systems. The Fc-tagged ECD of FZD6 (residues 19-132, Uniprot060353-1) was expressed and purified from Expi293 cells using the pFUSE-hIgGl-Fc2 vector (Invivogen) and the single protomer species was separated from aggregated protein by size exclusion chromatography on a Superdex 200 (10/300) column (GE Healthcare). Fe-tagged ECD fusion proteins of human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025/CF) were purchased from R&D Systems. WNT1 (5RP4754-1Oug), WNT26 (3900-WN-010/CF), WNT5a (645-WN-010/CF) and WNT3A (5036-WN-010/CF) were purchased from R&D Systems, and WNT3A conditioned media was prepared as described (PMID:12717451). Other proteins and chemicals were purchased from the following suppliers: FcRN (R&D, 8693-FC), C1q (Sigma, C1740), CD16a (R&D, 4325-FC), CD32a (R&D, 1330-CD/CF), CD64 (R&D, 1257-FC), LGK974 (Cayman Chemicals), the Porcupine Inhibitor C59 (Dalriada Therapeutics), and CHIR99021 (Sigma Aldrich).
3. Tetravalent binding molecules for FZD and LRP, "FLAgs", and antibody cloning 101961 DNA fragments encoding antibody (Ab) variable domains were either amplified by the PCR from phagemid DNA template or were constructed by chemical synthesis (Twist Biosciences). The DNA fragments were cloned into mammalian expression vectors (pSCSTa) designed for production of kappa light chains and human IgG1 heavy chains.
Bispecific diabodies and IgGs contained an optimized version of a "knobs-in-holes"
heterodimeric Fc (Ridgway et al. Protein Eng. 9, 617-621 (1996)). FLAgs and diabody-Fc fusions were arranged a VH-VL orientation with the variable domains separated by a short GGGGS (e.g. amino acids 121-125 of SEQ ID NO: 2) linker, which favors intermolecular association between VH and VL domains and thus favors diabody formation. To produce diabody-Fc fusion constructs, diabody chains were fused to human IgG1 Fc. FLAg proteins were constructed as VH-x-VL-y-[human IgG1 Fc1-z-VH-x-VL where linkers are x=
GGGGS
(e.g. amino acids 121-125 of SEQ ID NO: 2), y= LEDKTHTKVEPKSS (amino acids 245 of SEQ ID NO: 4), and z= SGSETPGTSESATPESGGG (amino acids 473 to 501 of SEQ
ID NO: 4). In this format, the human IgG1 Fc or knob-in-hole IgG1 Fc fragments spanned from position 234-478 (Kabat numbering). For scFv-Fc fusions, the variable domains were arranged in a VL-VH orientation and were connected by a long GTTAASGSSGGSSSGA
(SEQ ID NO: 75) linker, which favors intramolecular association between VH and VL
domains and thus favors scFv formation. For all constructs, the entire coding region was cloned into a mammalian expression vector in frame with the secretion signal peptide.
4. Protein expression and purification 101971 Antigen, Ab, and FLAg proteins were produced in Expi293F (ThermoFisher) cells by transient transfection_ Briefly, cells were grown to a density of approximately 2.5 x 106 cells/ml in Expi293 Expression Media (Gibco) in baffled cell culture flasks and transfected with the appropriate vectors using FectoPRO transfection reagent (Polyplus-transfection) using standard manufacture protocols (ThermoFisher). Expression was allowed to proceed for 5 days at 37 'V and 8% CO2 with shaking at 125 rpm. After expression, cells were removed by centrifugation and protein was purified from the conditioned media using rProtein A Sepharose (GE Healthcare). Purified protein was buffer exchanged into either PBS
or a formulated stabilization buffer (36.8 mM citric acid, 63.2 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0) for storage. Proteins concentrations were determined by absorbance at 280 nm and purity was confirmed by SDS-PAGE
analysis.
5. In vitro binding assays 101981 BLI assays were performed using an Octet HTX
instrument (ForteBio). For measuring binding to antigen, Fe-tagged fusions of FZD receptors (FZD-Fc proteins) were captured on AHQ BLI sensors (18-5001, ForteBio) to achieve a BLI response of 0.6-1 nm and remaining Fc-binding sites were saturated with human Fc (009-000-008, Jackson ImmunoResearch). FZD-coated or control (Fe-coated) sensors were transferred into 100 niv1 Ab or FLAg in assay buffer (PBS, 1% BSA, 0.05% Tween20) and association was monitored for 300 seconds. Sensors were then transferred into assay buffer and dissociation was monitored for an additional 300 seconds. Shake speed was 1000 rpm and temperature was 25 C. End-point response values were taken after 295 seconds of association time.
End-point data were analyzed by subtracting the Fe signal from the FZD-Fc signal and then normalizing the data to the highest binding signal.
[0199] For measuring binding to Fc receptors, Abs or FLAgs were immobilized on AR2G sensors (18-5092, ForteBio) by amine coupling to achieve a BLI response of 0.6-3 nm and remaining sites were quenched with ethanolamine. Coated sensors were equilibrated in assay buffer (PBS, 1% BSA, 0.05% Tween20) and transferred into Fc receptor solutions.
Association was monitored for 600 seconds, the sensors were transferred to assay buffer, and dissociation was monitored for 600 seconds. CD64 and all other Fc receptors were assayed at 50 n.M or 300 rtM, respectively, at pH 7.4, unless as indicated. Shake speed was 1000 rpm and temperature was 25 C. End-point response values were taken at the end of the association phase and were normalized to isotype controls. Steady-state FcRN
binding assays were performed in a similar manner, except that FcRN was immobilized and serial dilutions (0.1 ¨ 225 nM) of Ab or FLAg were assessed in solution. The association and disassociation times were 600 or 1200 seconds, respectively.
[0200] Surface plasmon resonance (SPR) assays were performed using a ProteOn XPR36 system (Bio-Rad). FZD-Fc or LRP-Fc proteins were immobilized to GLC sensor surface (176-5011) using standard amine coupling chemistry. Abs or FLAgs in assay buffer (PBS, 0.05% Tween20, 0.5% BSA) were injected at 40 ttl/min and association was monitored for 150 seconds. Assay buffer was then injected at 100 pl/min and dissociation was monitored for 900 seconds. Assays were performed at 25 C. Analysis was performed using a 1:1 Langmuir model and globally fit to determine kon and koff values using ProteOn Manager software. KD was calculated as the ratio of koff/kon.
6. Epitope binning [0201] BLI epitope binning experiments were performed using an Octet HTX instrument (ForteBio). Fc fusions with FZD ( FZD-Fc) or with LRP6 (LRP6-Fc) protein were immobilized on AHQ (18-5001, ForteBio) or AR2G (18-5092, ForteBio) BLI
sensors, respectively. Coated sensors were transferred into 100 nryl Ab in assay buffer (PBS, 1% BSA, 0.05% Tween20) for 240 seconds to achieve saturation of binding sites. Sensors were then transferred into 100 rtM competing Ab in assay buffer for 180 seconds.
Response at 20 seconds after exposure to competing Ab was measured and normalized to binding signal on unblocked antigen-coated sensors. Shake speed was 1000 rpm and temperature was 25 oC.
7. Cell lines [0202] HPAF-II and HEK293T cell lines were maintained in DMEM containing 4.5 g/L
D-glucose, Sodium pyruvate, L-glutamine (ThermoFisher #12430-054) and supplemented with 10% FBS (ThermoFisher) and Penicillin/Streptomycin (ThermoFisher 415140-163).
CHO cells were maintained in DMEM/F12 (ThermoFisher #11320-033) supplemented with 10% FBS and penicillin/streptomycin. Cells were maintained at 37 C and 5%
CO2.
8. Flow cytometry [0203] Indirect immunofluorescence staining of cells was performed with 10 n.M anti-FZD Fab for the CHO cell lines as previously described (Steinhart et al. 2017 Nat Med. Jan;
23(0:60-68, PMID: 27869803). Alexa Fluor 488 AffiniPure F(ab)2 was used as the secondary antibody (Jackson ImmunoResearch, 109-545-097). Anti-c-Myc IgG1 9E10 (primary antibody, ThermoFisher, MA1-980) and Alexa Fluor 488 IgG (secondary antibody, Life technologies, A11001) were used as controls for expression. All reagents were used as per manufacturer's instructions.

9. Luciferase reporter assay 102041 ILEK293T cells were transduced with lentivirus coding for the pBARls reporter (Biechele and Moon in Writ Signaling: Pathway Methods and Mammalian Models , E.
Vincan, Ed. (Humana Press, Totowa, NJ, 2008), pp. 99-110) and with Renilla Luciferase as a control to generate a Wnt-itcatenin signaling reporter cell line. 1-2 x 103 cells in 120 J4l were seeded in each well of 96-well plates for 24 hours prior to transfection or stimulation. The following day, FLAg or Ab protein was added, and following 15-20 hours of stimulation, cells were lysed and luminescence was measured in accordance with the dual luciferase protocol (Promega) using an Envision plate reader (PerkinEmer). For the FZD4-specific agonist assay, FZD4 cDNA was transfected for 6 hours prior to adding FLAg protein. For the Wnt inhibition assays, Wnt1 was introduced by cDNA transfection or WNT3A
protein was applied for 6 hours prior to the addition of Ab protein. All assays were repeated at least three times.

10. Western blot assay 102051 H1 ESCs were solubilized with lysis buffer (1%
Nonidet P40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 50 mM Tris (pH 7.4), 0.1 mM
EGTA, 0.1 mM EDTA, 20 mM sodium fluoride (NaF), 1:500 protease inhibitors (Sigma) and 1 mM
sodium orthovanadate (Na3V00). Lysate was incubated for 30 min at 4 C, centrifuged at 14,000 x g for 10 min, boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane and Western blotted using indicated Abs. Ab detection was performed by a chemiluminescence-based detection system (ECL; ThermoFisher).

11. Crystal violet proliferation assay 102061 HPAF-11 cells were seeded at 500 cells per well, and after 24 hours, 100 nIvl LGK974 was added with or without 100 nM FLAgi Medium was changed and drug treatment was renewed every other day. Cells were fixed with ice-cold methanol after 7 days treatment.
Cells were stained with 0.5% crystal violet solution in 25% methanol, destained in 10%
acetic acid and quantified by measuring absorbance at 590 nm.

12. Immunofluorescence 102071 H1 hES treated with FLAg and CH1R99021 for 3 days were washed with cold PBS, and fixed for 20 min with 4% PFA. Fixed cells were rinsed with PBS, pertneabilized with 0.3% triton for 10 min, and blocked with 1% BSA for 1 hour. Cells were incubated for 2 hours with primary Abs for BRACHYURY (R&D systems 4F2085; goat; dilution 1:100) or OCT3/4 (Santa Cruz sc5279; mouse; dilution 1:100 ) in 1% BSA and 1 hour with Alexa Fluor 488-labeled donkey anti-goat or Alexa Fluor 568-labeled donkey anti-mouse Ab (FIG.
13A). Coverslips were mounted using Fluoromount (Sigma-Aldrich) and analyzed on a Zeiss LSM700 confocal microscope using a 60x oil objective (FIG. 1313). Images were assembled using ImageJ and Photoshop CS6 (Adobe Systems, Mountain View, CA).

13. Intestinal crypt self-renewal assay 102081 8-10 week-old Lgr5-EGFP-IRES-creERT2 (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were performed according to protocols approved by the Animal Care and Use Committee at the University of Toronto, and complied with the regulations of the Canadian Council on Animal Care and with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments).
P P-L6 1+3 or a negative control Ab was reconstituted in 37 mM Citric Acid, 63 mM
Na2HPO4, 10% trehalose, 0.2M L-Arginine, 0.01% polysorbate 80, pH 6Ø The Porcupine Inhibitor C59 was reconstituted with 0.5% methylcellulose mixed with 0.1%
Tween 80 in ddH20. The mice (male and female) were divided into three groups (5-7 per group): vehicle, control (C59 and control Ab) or FLAg (C59 and F P+P-L6 1 3 On day 1, mice were treated by intraperitonea1 injection with vehicle, or 10 mg/kg control Ab or F'-L6'3. The treatments were blinded to the investigators until the end of the experiment and were repeated every two days for a total three treatments. Starting on day 2, vehicle or 50 mg/kg C59 was administered by gavage to the vehicle group or the two experimental groups, respectively, twice a day with 8 hours interval for 4 days. On day 6, the mice were sacrificed. The whole intestinal tissue was harvested, cleaned with cold PBS, dehydrated with PBS, 30% sucrose, fixed with 4% paraformaldehyde and embedded in optimal cutting temperature compound (OCT). 8 gm OCT frozen sections were used for immunohistology. The intestinal EGFP
crypts were analyzed using confocal microscopy (Zeiss LSM700). Representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with vehicle, C59 or pan-FLAg(FP+13-L61+3 ) + C59 are depicted in FIG. 14. LGR5-GFP is expressed in the stem cells at the bottom of crypts. Cell nuclei were counterstained with DAPI.
[0209] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein.
Such equivalents are considered to be within the scope of the inventions.
Various substitutions, alterations and modifications may be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention. The contents of all references, issued patents, and published patent applications cited through this application are hereby incorporated by reference. The appropriate component, process and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof C
0) I-a Ln N) N) No ID DNA
SEQ Protein SEQ
ID

ID 5 it GAGGITCAGCTGGIGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCACCTTCTGGCT

knob-TCGGTICTICTICTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATTIATTCTGCITT
TGCCTCTA SSSIHWVR QAPG KG LEWVASIYSAFASTSY

CECAGACACATCCAAAAACAC.AGCCTACCTACAAATGAACAGCTTA ACISV KG
RFTISADTSKNTAYLQMNSLRAED

CTTIGGACTACTGGGGICAAGGAACCCT
TAVYYCARYHFPFGFALDYWGQGTLVTVS
GGICACCETCTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGICCGCCTCTGTGGGC
GATAGG SGGGGSDI OMIQSPSSISASVGDRVTITCR
GTCACCATCACCTGCCGTGCCAGTCAGTCCGMTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAACCTCCGAA
GCTICT ASQSVSSAVAWYQQKP GKAPKWYSASSL

GATTTCACTCTGACCATCAG
TSGVPSRFSGSRSGTOFTLTISSLQPEDFAT
CAGTCTGCAG CCGGAAGACTTCGCAACTTATTACTGTCAG
CAAGGIGTTTACCTGTTCACGTTCGGACAGGGTACCAAGGTGGAG
YYCQQGVYLFTFGQGTKVEIKLEDKTHTKV
ATCAAACTCGAGgataaaactc.a co caAAAGTGGAGCCCAAAACTICTgatoagacccatacttgoccaCCGTGCCCAGCACCTGAACTCCTG
EPKTSDKTHTCPPCPAPELLGGPSVFLPPK
GGGGGACCGTCAGICTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCG
TGGIGGT PKDILMISRTPEVTCVVVDVS HEDPEVKFN
GGACGTGAGCCACGAAGACCCTGAGGICSIAGTTCAACTGGTACGTGGACGECGTGGAGGTGCATAATGCCAAGACAAA
GCCGCG WYVDGVEVHNAKTKPREEQYNSTYRWS
CGAGGAGCAGTACAACAGCACGTACCGTGIGGICAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAA
VLTVLHQDWLNGKEYKCKVSNKALPAPIE
esµ
GTGCAAGGICTCCAACAAAG
CCCTCCCAGCCCCCATCGAGAAAACCATerCCAAAGCCAAAGGGCAGCCCCGAGMCCAATGGIG
KTISKAKGQPREPMVFDLPPSREEMTKNQ
ITTGACCTE CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTG
GTGCATGGICAAGGGCUCTATCCCAG CGACA
VSLWCIVIVKG FY PSD lAVEWESNGQPEN N
TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACCCCTCCCGTGCTGGACTCCGACGE
CTCCTTCTT YKTTPPVLDSDGSFFLYSKLTV
DKSRWQQG
CCTGTACAGCAAGCTCACCGTGGACAAGAGCCGCTGGCAGCAGGCGAACGTUTCTCATGCTCCGTGATGCATGAGGCTC
TGCAC NVFSCSVM H EALHNHYTQKSLSLSPGKSG

CCACACCCG SETPGTSESATPESGGGEVQLVESGGGLV
AAAGTGETGGCGGAGAGGTICAGCTGGIGGAGTCTGGCGGTGGCCTEGTGCAGCCAGGGGGCTCACTCCGTITGTCCTG
TGCAG PG GSLRLSCAASGFN ISYSSIHWVRCIAPG
K
CTICTGGCTTCAACATCTMATTCTICTATCCACTGGGIGCGTCAGGCCCCGGETAAGGGCCTGGAATGGGITGCATATA
TTTM GLEWVAYISSYYGYTYYADSVKGRFTISADT
CITATTATGGCTATACTTATTATGCCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAAACACAGC
CTACCTAC SK NTAYLQM NSLRAEDTAVYYCARAHYFP
AAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCG
CTCATTACTTCCCGTGGGCTGGTGCTATGGACTAC WAGAM

TGGG
GICAAGGAACCCIGGICACCGTCTCCCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCC
G MTORSSLSASVGDRVTITCRASQSVSSAV
CCTCTGTGGGCGATAGGGICACCATCACCTGCCGTG CCAGTCAGTCCGTGTCCAGCGCTGTAG
CCTGGTATCAACAGAAACCAGG
AWYQQKPGKAPKLLIYSASSLYSGVPSRFS

CGGA GSRSGTD FTLTISSLQP EDFATYYCQQrfW
TTTCACTCTGACCATCAG CAGTCTGCAGCCGGAAGACTICGCAAMATTACTGTCAG

GGGTACCAAG GTGGAGATCAAA
No en C
0) I-a Ln N) N) TABLE 1.A

No _ .
ID DNA
SEQ Protein SEQ
ID

ID

CAGCCAGGGGGCTCACTCCGTTIGTCCTGTGCAGCTICTGGCTTC¨AA CA 3 ho TCGGITCTICTICTATC CA CTG GGTG CGTCAGG CCCCG GG TAAG GG
CCTG GAATG G GTTGCATCTATTTATTCTGCUTTG C CTCTA EVQLVESGGGLVQPGGSLRLSCAAK FN
IG

CGCAGACACATCCAAAAACACAGCCTACCIACAAATGAACAGCTTA
SS I HWVROAPG KGLEWVASIYSAFASTSY

CTCGCTACCATTTCCCGTTCGGTITTGCTTTGGACTACTGGGETCAAGGAACCCT
ADSVKGRFTISADTSKNTAYLQMNSLRAED
GGTCACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGICCGCCICTGIGGGC
GATAGG TAVYYCARYHFP FGFALOYWGCLGTLVTVS
GICACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTG
TAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCT
SGGGG SDI QMTQSPSSLSASVGDRVTITCR
GA1TrACTCG6 CATCCAGCCTCTACTCTG GAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCG
GGACGGATTTCACTCTGACCATCAG
ASQSVSSAVAWYQQKPGKAPKWYSASSL
CAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGICAGCAAGGTGTTTACCTGTTCACGTTCGGACAGGGTACCAAG

FSGSRSGTDFTLTISSLQPEDFAT
ATCAAACTC6AGgacaaaactcacacaAAAG1TGAGCCCAMTCTTCTgataagacccataatTGcccAcc6rGcccAGc AccTGAAcTccT YYCQQGVYLFTFG QGTKVEIKLEDKTHTKV
GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGG E PKSSDKTH NCPPCPAPELLGG PSVFLF
PPK
TGGACGTGAGCCACGAAGACCCTGAGGICAAGTMACYGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CG C PKDTLM ISRTPEVICVVV DVS
REDPEVKFN
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACA WYVDGVEVHNAKTKPREEQYNSTYRVVS
AGTGCAAGGICTCCAACAAAGCCCICCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGT

GTACACCCIGCCCCCAATCCGGGAGCTGATGACCAGCAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCC
AGCGAC KTISKAKGQPREPQVYTLP PIRE LM TS
N QV
ATCGCCGTGGAGIGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT
CCTICT SLSCAVKG FYPSDIAVEWES NGQPENNYK
TCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC

CAACCACTACACGCAGAAGAGCCICTCCCTOCTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCG
CCACACCC VFSCSVM HEALHN
HYTOKSISLSPEKSGSE
GAAAGTGGIGGCGGAGAGGTTCAGCTGGTGGAGTCTGGCEGTGG
CCTGGTGCAGCCAGGGGGCTCACTCCGTTIGTCCIGTE CA
TPGTSESATPESGGGEVQLVESG GGLVQP
GCTTCTGGCTTCAACATCTCTICTTATTATATCCACTGGGIGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGITGCAT
CTATTTAT GGSLRLSCAASGFNI SSYYIHWVROAPGKG
TCTICTTATGGCTATACTICTTATG CCGATAG
CGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTA
LEWVASYSSYGYTSYADSVKGRFTISADTS
CAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACT
ICTCTGG KNTAYLQMNSLRAE DTAVYYCARIVRGSK
TTGG G CTATGGACTACTGGGGICAAGGAACCCTGGICAC
CGTCTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTCC
KPYFSGWAM DYWGQGTLVT1/5.56GGGS
CCGAG
CTCCCTGTCCGCCTCTGTGGGCGATAGGGICACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGG
TA DIQMTQSPSSLSASVG DR VTITCRASQSVS
TCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCG G
CATCCAGCCICTACTCTGGAGTCCCTICTCGCTTCTCTGGTAG
SAVAWYQQKPG KAP KLINSASSLYSGVPS
CCOTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAG
CCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTCTTGGG

GTCCGTICACGIITCGGACAGGGTACCAAGGTGGAGATCAAA
SWGPFTFGQGTKVEI
No ea.
tit C
0) I-a Ln N) N) I-a TABLE IA

No ID DNA
SEQ Protein SEQ
I D

ID 5 it Fc- TCGGTTCTTCTTCTATCCACTGGGTG
CGTCAGGCCUGGGTAAGGGCCTGGAATGGGTTGCATCIATTTATTCTGCTTTTGCCTCTA
SSSIHWVR OAPG KG LEWVASIYSAFASTSY

CTICTTATGCCGATAGCGTCAAGGGCCGTTICACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAA
CAGCTTA ADSVKGRFTISADTSKNTAYLQMNSLRA ED
AGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTACCATTTCCCGTTCGGTTTTECTUGGACTACTGGGETCAAGG
AACCCT TAVYYCARYHFPFGFALDYWGQGTLVTVS
GGTCACCGTCTCCTCGG GTGGAG
GTGECAGTGATATCCAGATGACCCAGICCCCGAGCTCCCTETCCGCCTCIGTGGG CGATAGG
SEG GGSDI QMTQSPSSLSASVGDRVTITCR
GTC.ACCATCACCTGCCGTGCCAGTCAGTCCGTGTCC.AGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCC
GAAGCTTCT ASQ$V$$AVAWYQQKPGKAPKLLIYSASSL
GATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTICTCGCTICTCTGGTAGCCGTTCCEGGACGGATTTCACTCTG
ACCATCAG YSGVPSRFSGSRSGTDFTLTISSLQP
EDFAT
CAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGIGTTTACCTOTTCACGTTCGGACAGGGTACCAAG
GTGGAG YYCQQGVYLFTFGQGTKVEI KLEDKTHTKV
ATCAAACTCGAGga ca a a actocacaAAAG TTGAG C
CCAAATC17CTgataagacccatacttgcccaccgtgc c ca g ca cctga a ctcctgggigga cc EPKSSDKTHTCPPCPAPELLGGPSVFLFPPK
gtca gtatcctettcccccraa a a ccca ag gaca ccctcatgatctcccg gacccctga ggtcacatgcgtggtgeggacgtgagccacgaagaccctgaggtcaagtt P KDTLM ISRTP
EVTCVVVDVS HE DPE V KFN
ea actggtacgtggacggcgtggaggtgcataa two aga ca a agccgagggaggagcagta ca ca gca cgtaccegtEgtcagcgtcctca ccgtcctgcaccag WYVDGVEVHNAKTKP REEQYNSTYRVVS
ga ttlictga atgg ca a gga gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga gaaccacag VLIVLHQDWLNGKEYKCKVSN KALPAPI E
gtgta caccotgcccccatcccgggagga ga tga ccaa g aaccaggtcagcctga cctgcctggtca a a guttctatcccapgacatcgccgtegagtegga ga gcaa KTISKAKGQPREPQVYTLP PSREEMTKNQ
tgggCacteggagea caactacaagaccacgcctacgtgaggactccgategctcctIcttcctcta cagna gctca agtggaca agagcaggtggc a gcaggeg VSLTCLVKGFYP SD lAVEWES
NGQPE N NY
aacgtottctcatgctccgtgetgcacgaggctctgca ca a ccacta ca cgca gaag agcctaccctgtctccgutaa a AGCGGCAG CGAGACTCCCGGGAC
KTIPPVLDSDGSFFLYSKLTVOKSRWQQG

GIGGCCTEGTGCAGCCAGGGGGCTC NVFSCSVMHEALH

ACTCCGTTIGTCCTGTGCAGCTTCTGGCTTCAACATCTCTIATTCTTCTATCCACTGGGTG
CGTCAGGCCCCGGGTAAGGGCCTGGA SET
PGTSESATPESGGGEVQLV ESGGGLVQ
ATGGGITGCATATATTTCTTMATTATGGCTATACTTATTATGCCGATAG
CGICAAGGGCCETTrCACTATAAGCGCAGACACATC
PGGSLRLSCAASGFN ISYSSIHWVRCIAPGK
CAAAAACACAGCCTACCTACAAATGAACAG
CTTAAGAGCTGAGGACACTGCCGICTATTATTGTGCTCGCGCTCATTACTTCCCGT
GLEWVAYISSYYGYTYVADSVKGRFTISADT

GTCAAGGAACCCTGGICACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCC
SKNTAYLQMNSLFtAEDTAVYYCARAHYFP
AGTCCCCGAGCTCCCTGTCCGCCTCTGTGGG
CGATAGGGICACCATCACCTOCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAG CC
WAGAM DYWGQGTLVTVSSGGGGSD I Q
TEGTATCAACAGAAACCAGGAMAGCTCCGAAGCTICTGA'TTTACTCGGCATCCAGCCICTACTCTGGAGTCCC7TCTC
ECTTCTCT MTQSPSSLSASVGDRVTITCRASQSVSSAV
GGTAG
CCGTTCCGGGACGGATTICACTCTGACC.ATCAGCAGICTGCAGCCGGAAGACITCGCAACTTATTACTGTCAGCAATA
CTC AWYQQKPGKAPKWYSASSLYSGVPSRFS
TTGGG GTCCGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA
GSRSGTD FTLTISSLQPEDFATYYCQQYSW
GPFTFGQGTKVE I K

No C
0) I-a Ln N) N) TABLE lA

No ID DNA
SEQ Protein SEQ
ID

GGLVQPGGSLRLSCAASGFNIG
Fc-GCCTCTA SSSI HWVROAPGKGLEWVASIYSAFASTSY

CGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCITA
ADSVKGRFTISADTS KNTAYLQM NSLRAED
AGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTACCATTTCCCGTICGGTTTTGCTTTGGACTACTGEGGTCAAG
GAACCCT TAVYYCARYHFPFGFALDYWGQGTIVIVS
GGICACCGTCTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCIGTGGGC
GATAGG %GEC SVCIMIQSPSSLSASVGDRVTITCR
GTCACCATCACCIGCCGIGCCAGICAGTCCGTGTCCAG CGCTGTAGCCTGGTATCAACAGAAACCAG
GAAAAGCTCCGAAG CTTCT
ASQ,SVSSAVAWYQQKPGKAPKWYSASSL
GATTTACTCGGCATCCAGCCICTACTCTGGAGTCCCTTCTCGCITCTCTGETAGCCETTCCGGGACGGATTICACTCTG
ACCATCAG YSGVPSR FSGSRSGTDFTLTISSLQPEDFAT
CAGTCTGCAGCCGGAAGACTTCGCPACTTATTACTGTCAG CAAGGTG TTTA C CTGTTCACGTTCG
GACAGGGTA C CAA GGTGGAG
YYCQQGVYLFIFGQGTKVEIKLEDKTHTKV
ATCAAACTCGA Gga ca a a a ctca cacaAAAG TTGAG CCCAAATCTTCTga ta a pccc atacttucca c cgtgcccagca cctgaactc ctgggggga cc EPKSSDKTHTCPPCPAP
ELLGGPSVFLFPPK
gtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg gacgtgagccacgaagaccctgaggtcaagtt PKDILMISRTPEVICVVVDVS HE DP
EVKFN
caactggtacgtggacggcgtggaggtgcataatgccaaga C8 aagccgcugaggagcara ca 8 ca gca cet a ctegtggtca gcgtatcaccecctsca cca g WYVDGVEVH NAKT KP RE
EQYNSTYRVVS
ctµ
gactggctgaatgecaauagtacaagtraaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa gccaaagggcagccccgagaaccacag VLTVLHQDWLNGK EY KCKVSN KAL PA PI E
gtgtaca ccctgcccccatcccggpggagstgaccaagaaccaggtca gcctgacctgcct.ggtcaaaggcttctatcccagcga catcgccgtggagtggga ga gca a KTISKAKGQPREPQVYTIPPSREEMTKNQ
tgggcagccggaga aca a ctecaagaccacgcctcccgtgctggactccgacggctccttcttcctcta cagcaagetcaccgtggacaagagcaggtggcagcagggg VSLTCLVKGFYPSDIAVEWESNGQPE
N NY
aacgtcttctcatgaccgtgatgtacgaggctctgcacaa ccactacacgcagaagagcctctccctgtctccg,ggtaaaAGCGGCAGCGAGACTCCCGGGAC
KTTPPVLDSDGSFFLYSKLIVOKSRWQQG
CTCAGAGTCCGCCACACCCGAA.AGIGGTGGCGGAGAGGTICAGCTGGIGGAGTCTGGCG

EALHN HYT QKSLSLSPG K SG
ACTCCGTITGTCCIGTGCAGCTTCTGG CTTCAACATCTCTTCTTATTATATCCACTGGGTG
CGTCAGGCCCOGGGTAAGGGCCTGGA
SETPGTSESATPEGGGEVOLVESGGGLVQ
AIGGGITGCATCTATTTATICTTCTTATEGCTATACTICTTATGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCA
GACACATC PGGSLRLSCAASGFN ISSYY I
HWVRQAPEK
CAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGITCGT
GGATCCA C LEWVASIYSSYGYTSYADSVKGRFTISADT
AAAAACCGTA ClICTCTG GTTGGGCTATGGACTA CTGGGGTCAAGG AA CCCTGG TCACCGICTCCTCG
GGIG GAGGIGGCAGTGA SKNTAYLQM
NSLRAEDTAVYYCARTVRGS
TATCCAGATGACCCAGTCCCCGAGCTCCCTGICCGCCTCTGTGGGCGATAGGGTCACCATCACCTG C
CGTGCCAGTCAGTCCGTGT KKPYFSGWAMDYWG
QGTLVTVSSGGG G
CCAGCGCTGTAGCCTG GTATCAACAGAAACCAGGAAAAG
CTCCGPAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTC

CCITCTCGCTTCICTEGTAGCCGTTCCGGGACGGATTTcAcTcTGACCATCAGCAGTCTGCAGCCGGAAGACTICGCAA
CTTATTAC SSAVAWYQQKPGKAPKLUYSASSLYSGVP
TGTCAGCAATACTACTGGCCGATCACGTTCGGACAGGGTACCAAGGIGGAGATCAAA

EDFATYYCQQ
YYWPITFGQGTKVEIK

No ea.
=

C
0) I-a Ln N) N) No ID DNA
SEQ Protein SEQ
ID

ID ot knob-TGGCTATA YYYMHWVR OAPGKG LEWVASIYSYYGYT

CAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
YYADSVKG RFTISADTSICNTAYLQMNSLRA

EDTAVYYCARSSFSWAM DYWGQGTLVTV
CGTOICCICGGGIGGAG GTGGCAG TGATATCC.AG ATGAC CCAGTCCCCGA GCT CCCTG TCCG
CCTCTGTGGGCGATAGGGTCACC SSG
GGGSDIQMTQSPSSLSASVGDRVTITC

CCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTICTGATTTA
RASQSVSSAVAWYQQKPGKAPKLLIYSASS
CTCGGCAICCAGCCTCTACTCTGGAGTCCCTICTCECTTCTCTGGTAGCCGITCCGGGACGGAITTCACTCTGACCATC
AGCAGTCT LYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT
GCAGCC.GGAAGACTICG

GTG 6AGATCAAACTCGAGgacaa a actcacacaAAAGIGGAGCCCAAAACTICTgataagacccatactTG
CCCACCGTGCCCAGCACCTG HTKVEPKTSDKTFITCPPCPAP ELLGG
PSVF
AACTCCTGGG

LFPP KPKDTLMISRTP EVTCVVVDVSHE DP

CGTGGAGGTGCATAATG C CAAGAC A
EVKFNWYVDEVEVNNAKIKPREEQYNST
ctµ AAGCCGCGCGAGGAGCAGTACAACAGCACGTACCGTGTG
GICAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG YRVVSVLTVLH
QOM G KEYKCKVSN KAL
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCICCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAAC PAPI E KTISKAKG QP R EPM V
FDLPPSREEM

AACCAGGTCAGCCIGTGGTGCATGGICAAGGGCTTCTATCC
TKN QVSLWCMV KGFYP SDI AV EW ESNG Q

LDSOGSFFLYSKLTVD KSR

ATGAGG WQQGNVFSCSVMHEALHNHYTQl(SLIS
CTCTGCACAACCACTACACGCAGAAGAGCCICICCCIGTCTCCG GGTAAAAGCGGCAG
CGAGACTCCCGGGACCTCAGAGTCCGC
PGIGGSETPETSESATPESGGGEVQLVESG
CACACCC.GAAAGTEGIGGCGGAGAGGITCAGCTGGIGGAGTCIGGCGGTGGCCIGGIGCAGCCAGGGGGCTCACICCG
TTTGIC GG LVQPGGSLRLSCAASGFNISYSSIHWVR
CTGTGCAGCTICTGGCTTCAACATCICTTATICTICTATCCACTGGGTGCGTCAG G CCCCG
GGTAAGGGCCTGGAATGGGTTG CAT
QAPGKGLEWVAYISSYYGMYADSVKGRF
ATAMCTTCTTATTATGGCTATACTTATTATGCCGATAG
cGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAG
TISADTSKNTAYLQM NSLRAEDTAVYYCAR
CCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTTCCCGTGGGC
TGGTGCT Al-IYFPWAGAMDYWGQGTINTVSSGGEG
ATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACC
CAGTCCCCGAGCT
SDIQMTQSPSSLSASVGDRVTITCRASQSV
CCCTGT CCGCCTCTGTGGGCGATAG GGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCG
CTGTAGCCTGGTATCAACAG SSAVAWYQQKPGKAPK
LINSASSLYSGVP
AAACCAGGAAAAGCTCCGAAECTTCTGAMACTCGGCATCCAGCCTCTACTCTGGAGTCCCITCTCGCTICTCTGGTAG
CCGTTCC SRFSGSRSGTO FILTISSLQPEDFATYYCQQ
GGGACGGATTTCACICTGACCATCAGCAGICTGCAGCCGGAAGACTTCGCAACTrATIACTGICAGCAATACTACIGGC
CGATCAC YYWPITFGQGTKVEIK

GTTCGGACAG GGTACCAAGGTGGAGATCAAA
No C
0) I-a Ln N) N) TAB LE 1.A

No ID DNA
SEQ Protein SEQ
I D

ID 5 it CTGGTGGAGTCTGGCGGTGGCCTGGIGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACA

hole- TCTCTTATTATTATATG CACTGGGTGCGTCAGGCCCCGG GTAAGGG CCTGGAATG
GGTTGCATCTATTTATTCTTATTATG GCTATA
YYYMHWVRCIAPGKGLEWVASIYSYYGYT

CCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
YYADSVKG RFTISADTSKNTAYLQMNSLRA

AGAGCTGAGGACACTGCCGTCTATTATTGTECTCGCTCTTCTTTCTUTGGGCTATGGACTACTGGGGICAAGGAACCCT
GGTCAC EDTAVYYCARSSFSW AM DYWGQGTLVTV
CGICTCCTCGGGTGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGG
GTCACC SSG GGGSDIWATQSPSSLSASVG
DRVTITC
ATCACCIGCCGTGCCAGTCAGTCCGTEICCAGCGCTGTAGCCIGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTC
TGATTTA RASQSVSSAVAWYQQKPGKAPKWYSASS
CTCGGCATCCAGCCTCTACTCTGGAGTCCCTICTCGCTTCTCTG G TAG CCGTTC CGE GACG G
ATTTCACTCTGAC CATCAG CAGT CT
LYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT
GCAGCCGGAAGACTTCGCAACTTATTACTGICAGCAACATCCGTGGTCTGGTGGTTACCTGATCACGTTCGGACAGGGT

KT
GTGGAGATCAAACTCGAGga caa ea ctoca ca AAAGTTGAG CCCAAATCTTCTga ta agacccataatTECCCACCGTOCCCAGCACCTGA
HTKVEPICSSDKTHNCPPCPAPELLGGPSVF
ACTCCTGGGGGGACCGTCAGTCTTCCTCTECCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGIC
ACATGCG LFP PKPKDTLM ISRTP EVTCVWDVSH
EDP
TGGTGGIGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAA EVICFNINYVDGVEVHNAKTK PREEQYNST
esµ
AGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGG YRVVSVLTVLHQDWLNGKEYKC KVSNKAL
AGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC.ATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACC PAPIE KTI SKAKGQPR
EPCNYTLPPIRELMT
ACikGGTGTACACCCTG
CCCCCAATCCGGGAGCTGATGACCAGCAACCAGGICAGCCTGAGCTGCGCCGTCAAAGGCTICTATCCC
SNQVSLSCAVKGFYPSDIAVEWESNGQP E
AG CGACATCGCCGTG GAGTGG GAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG G C
NNYKTIPPVLDSOGSFFLVSKLTVDKSKW
TCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGETGGCAGCAGGGGAACGICTTCTCATGCTCCGTGATGC
ATGAGE QQGNVFSCSVMHEALHNHYTOOLSLSPG
aCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAG
TCCGC KSGSETPGISESATPESGGGEVQLVESGGE
CACACCCGAAAGTGGIGGCGGAGAGGTTCAGCTGGIGGAGTCTGGCGGTG G CCTG G TG
CAGCCAGGGGGCTCACTCCGITTGIC
LVQPGGSLRLSC.AASGFN ISSYYIHWVROA
CIGTGCAGCTICIGGCTTCAACATCTCTICTTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGG
GTTGCAT PGKGL

CTATTTATTCTICTTATGGCTATACTTCTTATOCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCC.A
AAAACACAG ADTSKNTAYLQM NSLRAEDTAVYYCARTV
CCTACCTACAAATGAACAGCTTAAGAGCTGAG

RGSKKPYFSGWAM DYWGQGTLVTVSSG
TTCTCTGGITGGGCTATGGACTACTGGGETCAAGGAACCCTGETCACCGTCTCCTCGGGTGGAGGTGGCAGTGATATCC
AGATGA GGGSDIQMTQSPSSLSASVGDRVTITCFtAS
CCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGICACCATC.ACCTGCCGTGCCAGTCAGTCCGTGICCA
GCGCTGTA QSVSSAVAWYQQKPGKAPKWYSASSLYS
GCCTGGTATCAACAGAAACCAGGAAAAG CTCCGAAGCTTCTGAMACTCGGCATCCAG
CCTCTACTCTGGAGICCCITCTCUCTTC
GVPSRFSGSRSGTDFILTISS LCtPEDFATYY
TCTGGTAGCCGTTCCGGGACEGATTICACTaGAccxrc.AGcAGRTGCAGCCGGAAGACTTCGCAACTTATTACTGICA

CICTTEGGGICCGTTCACGTTCGGACAGGGTACCAAGGIGGAGATCAAA
No en C
0) I-a Ln N) N) I-a TABLE IA

ID DNA
SEQ Protein SEQ
ID

ID 5 it CAGCTICTGGCTICAACC 13 EVQLVESG GGLVCtPGGSLRLSCAASGFN LS 14 knob-TCTCTICTTATICTATGCACTGGGIGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATATATTTCTrCTTATTA
TGGCTATA SYSM HWVROAPGKG LEWVAYISSYYGYT

CCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
YYADSVKGRFTISADTS KNTAYLQM NS LRA

CTCGCCCGGCTCCGGGICATTGGGGTTTTGACTACTGGGGICAAGGAACCCTGG
EDTAVYYCARPAPG HWGFDYWGQGTLV
TCACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGIGGGCGA
TAGGGT TVSSGGGGSDIQMTO$PSSLSASVGDRVT1 =
CACCATCACCTG CCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTG GTATCAACAGAAACCAGGAAAAG
CTCCGAAGCTTCTGA TCRASCtSVSSAVAW YQQK PG
KAP laLlYSA
TTTACTCGG CATCCAG CCTCTACT CTG G AGTCCUTCT CGCTTCTCTG GTA GC CGTTC CG OG ACG
GATTTCA CTCTGACC ATCAG CA SSLYSGVPSRFSGSRSGTDFTLTISSLQP
EDF

CGTTCG GACAGG GTACCAAGGTG GA ATYYCQQWYYAPITFGQGTKV El KLEDKTH
GATCAAACCGAGgacaaa actcacecaAMGTGGAGCCCAAMCTTCTgataagacccatactTGCCCACCGTcCCCAGCACCTGMcTcc TKVEPKTSDKTHTCPPCPAPELLGGPSVFLF
TGGGGEGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATG
CGTGGTG P PK PKDTLM
ISRTPEITTCVWDVSHEDPEV
GTEGACGTGAGCCACGAAGACCCTGAGGICAAGITCAACTGGTACGTGGACGGCGTGGAGGIGCATAATGCCAAGACAA
AGCCG KFNWYV DGVEVHNAKTKP R E EQYN
STYR
CGCGAGGAGCAGTACAACAGCACGTACCETGIGGTCAGCGTCCTCACCGTCCTE
CACCAGGACTGGCTGAATGGCAAGGAGTAC
VVSVLIVLHQDW LNG KEYKCKVSNKALPA
oo AAGTGC.AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCAATGG PI EKTISKAKGQPREPMVFDLPPSREEMTK
TGTTTGACCTSCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGICAGCCTGTGGTGCATGGICAAGGGCTTCTATCC
CAGCGA NQVSLWCMVKGFYRSDIAVEWESNGQPE
cATcGcCGTeGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTC NNYKTTPPV LDSDGSFFLYSKLTVDKSRWQ
TTCCTETACAG CAAGCTCAC CGTG G A CAAGAG CCGCTG G CAG CAGGG GAACG TCTTCTCATG
CTCCGTG ATGCATG AG G CTCTGC QGNVFSCSV M H EA LH
NHYTQICSULSPGK
ACAACCACTACACGCAGAAGAGCCTCTCCCTGICTCCGGGIAAAAGCGG
CAGCGAGACTCCCGGGACCTCAGAGTCCG CCACACC
SGSETPETSESATPESGGG EVQLVESGGGL
CGAAAGTGGIGGCGGAGAGGTTCAGCTGGTG
GAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGC
VCtPGGSLRLSCAASG FNISYSSIHWVROAP
AGCTTCTGGCTTCAACATCTCTTATTCTICTATCCACTGGGIGCGICAGGCCCCGGGTAAGGGCCTGGAATGGGTTG
CATATATTTC GKGLEWVAYISSYYGTIVYADSV KGR
FT] SA
TTCTTATTATGGCTATACTTATTATG CCGATAG CGTCAAGGG CCGTTICACTATAAG
CGCAGACACATCCAAAAACA CAG CCTAC CT DISKNTAYLQM
NSLRAEDTAVYYCARAHY

TGGACT FPWAGAMDYWGQGTLVTVSSGGGGSDI
ACTGGG STCAAGG AACCCTGGTCACCGTCTCCTCGG GIGGAGGIG CAGTGATATCCAGATGACCCAGTCCCCG
AGCTCCCIGTC QMTQSPSSLSASVGDRVTITCRASQSVSSA
CGCCTCTGTGGGCGATAGGGTCACCATCACCMCCGTGCCAGICAGTCCGTGTCC.AGCGCTGTAGCCIGGTATCAACAG
AAACCA VAWYQQKPGKAPKWYSASSLYSGVPSRF
GGAAAAGCTCCGAAGCTTCTGATTIACTCGGCATCCAGCCTCTACTCTGGAGTCCMCTCGCTTCTCTGGTAG
CCGTTCCGGGACG SGSRSGTD
FTLTISSLQFEDFKFYYCQQYY
GATTTCACTCTGACCATCAG CAGTCTGCAG CCGGAAGACTTCG CAACTTATTACTG
TCAGCAATACTACTSGCCGATCACGTTCGG

ACAG G GTACCAAGGTGGAGATCAAA
= co S-D

C
0) I-a Ln N) N) No ID DNA
SEQ Protein ¨SECt ID

ID ot hole- TCTCTTCTTATTCTATGCACTGGGTSCGICAG G C CCCG G GTAAGG GCMG
AATGGGTTGCATATATTICITCTTAriATGGCTATA SYSMHWVROAPGKG LEWVAYISSYYGYT

CGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA YYADSVKG R FrISADTSKNTAYLQM
NSLRA
2542 AGAGCTGAGGACACTGCCGTCTATrATrGIG
CrCGCCCGGCTCCGGGICATTGGGGTTTTGACTACIGGGGTCAAGGAACCCTGG
EDTAVYYCARPAPGHWGFDYWGQGTLV
TCACCGTCTCCTCGGGIGGAGGTGECAGTGATATCCAGATGACCCAGICCCCGAGCTCCCTGTCCGCCTCTETGGGCGA
TAGGGT TVSSGGGGSDIQMTQSPSSLSASVGDRVTI
CACCATCACCTECCGTGCCAGTCAGTCCGTGTOCAGCGCTGTAGCCIGGTATCAACAGAAACCAGGAPAAGCTCCGAAG
CTTCTGA TCRASQSVSSAVAWYQQKPGKAPKWYSA
TTTACTCGG CATCCAGCCTCTACTCTG GAGTCCCIT CTCGCTICTCTGGTAG CCGTTCCG
GGACGGATTTCACTCTGACCATCAGCA SSLYSGVPSR FSGSRSGTDFTLTISSLQPEDF
GTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATGGTACTACGCTCCGATCACGTTCGGACAGGGTACCAA
GGIGGA ATYYCQQWYYAPITFGQGTKVEI KLEDKTH
GATCAAACTCGAGga caaaactcaca caAAAGTTGAGCCCAAATCTTCTgataagacceataatTGCCCACCGTGCCCAGCACCTGAACTCC
TKVEPKSSDKTHNCPPCPAPELLGGPSVFLF
TGGGGGGACCGTCAGICTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTG PPKPKDTLM ISRTPEVTCVWDVSHEDPEV

GAGGTGCATAATGCCAAGACAAAGCCG KFNWWDGVEVH NAKTK PR EEQY NSTY R
CGG GAGGAGCAGTACAACAGCACGTACCGTGTGETCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTAC VVSVLIVLHCIDWIN GKEYKCKVSNKALPA
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAG CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG
CCCCGAGAACCACAGG PIEKTISKAKGQPREPQVYTIPPIRELMTSN
TGTACACCCTGCCCCCAATCCGGGAGCTGATGACC.AGCAACCAGGTCAGCCTGAGCrG CGCCGTCAAAGG
CTTCTATCCCAGCGAC QVSLSCAVKGFYPSD !AVE WESNGQ.PEN N
ATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCT YKTTPPVLDS DGSFFLVSK LTV() KSRWQQ
TCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCTECA GNVFSCSV MHEALHN

CAACCACTACACGCAGAAGAGCCICTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCC
ACACCC GSETPGTSESATPESG GGEVQLVESGGGLV
GAAAGTGGTGGCGGAGAGGITCAGCTGGTGGAGTCTGG CGGIGGCCTEGTGCAGCCAGGGGG

GCTICIGGCTCAACATCTCTICTTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGITGCATC
TATTTAT KGLEWVASIYSSYGYTSYADSVKGRFTISAD
TCTTCTTATGGCTATACTTCTTATG CCGATAGCGTCAAGGGCCGTTTCACTATAAGCG
CAGACACATCCAAAAACACAGCCTACCTA TSKNTAYLQM NSLRAEDTAVYYCARTVRG
CAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACT
TCTCTGG SKK PYFSGWAM DYWGQGTLVTVSSGGG
TTGG
GCTATGGACTACTGGGETCAAGGAACCCTGGTCACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGT
CC GS DI QMTQSPSSLSASVG DRVTITCRASQS
CCGAGCTCCCIGTCCGCCTCTGTGGGCGATAGGGICACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAG
CCTGGTA VSSAVAWYQQKPG KAPKWYSASSLYSGV
TCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAG
CaCTACTCTGGAGTCCCFCCGCTTCTCTGGTAG PSRFSGSRSGTOFTLTISSLQPEDFATYYCQ
CCGTTCCGGGACGGATTICACTCTGACCATCAGCAGTCTGCAGCCGGAAGAMCGCAACTTATTACTGICAGCAATACTC
TIGGG QYSWGPFTFGQGTKVE I K

GICCGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA
No en C
0) I-a Ln N) N) TABLE IA

No ID DNA
SEQ Protein SEQ
ID

ID 5 it knob- TCT CTTATTATTATATGCACTGGGTGCGTCAGG
CCCCGGGTAAGGGCCTGGAAIGGGTTGCATCTATITCTTCTTATTATG G CTCTA
YYYMHWVR QAPGKGLEVVVASISSYYGST

CITATTAIGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAA
CAGCTTA TYADSVKG R FTISADTSKNTAYLQMNSLRA

AGAGCTGAGGACACTGCCGTCTATTATTGIGCTCGCTCTTGGIGGGCTTGGGCTTITGACTACTGGGGICAAGGAACCC
IGGICAC EDTAVYYCARSWWAWAFDYINGQGTLVT
CGTCTCCTCGGGTOGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCICIGIGGGCGAIAG
GGTCACC VSSG GGGSD I
CINITQSPSSISASVGDRVTIT
ATCACCTGCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAACCIIC
TGATTTA CRASO$VSSAVAWYQQKPGKAPKLLIYSAS
CTCGG
CATCCAGCCICTACTCTGGAGICCCTICTCGCTICICIGGTAGCCGTTCCGGGACGGATITCACTCTGACCATCAGCAG
ICT SLYSGVPSRFSGSRSETDFTLTISSLQPE DFA
GCAGCCGGAAGACTTCGCAACTTATTACIGTCAGCAACATTACTCTGTTTACGCTTCTCTGATCACGTTCGGACAGGGT
ACCAAGG TYYCQQHYSVYASLITFGQGTKVEIKLEDKT
TGGAGATCAAACTCGAGga caaaactca caca AAAGTGGAGCCCA AAACTTCTgata a gacccata ctIG
CCCACCGTG CCCAGCACCTGAA HTKVEPKTSDKTHTCPPCPAPE LLGG
PSVF
CTCCTGGGGGGACCGTCAGTCTICCTCTICCCCCCAAAACCCAAGGACACCCICATGATCTCCCGGACCCCTGAGGICA
CATGCGT L FP PKPIOTL MI SRIP
EVTCVVVDVSHE DP
GGIGGTGE ACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGIGGACGGCGIGGAG
GIGCATAAIGCCAAGACAAA EVITNWYVOGVEVH NAKTK
PRE EQYN ST
GCCGCGCGAGGAGCAGTACAACAGC.ACGTACCGTGTGGICAG
CGTCCTCACCGTCCTGCACCA,GGACTGGCTGAATGGCAAGGA
YRVVSVLTVLH QDWLN G KEYK CKVSNKAL
GTACAAGTGCAAGGTCTCCAACAAAG CCCTCCCAGCCCCCATCGAGAAAACC.ATCTCCAAAG CCAAAGG
GCAGCCCCGAGAACCA
PAPIEKTISKAKGQPREPMVFDLPPSREEM

ATCCCA TKNIQVSLWCMVKGFYPSDIAVEW ESN GO
GCGACATCGCCGTGGAGTOGGAGAG CAM GGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTECTGGACTCCGACEG CT PEN NY
KTTPPVL DSDGSFFLYSK LTVDKSR
CCTICTICCTGTACAGCAAGCTCACCGTGGACAAGAGCCG
CTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
WQQGNVFSCSVMHEALHNHYTOKSLSLS
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAGCGGCAGCGAGACTCCCGGEACCTCAGAGT
CCGCCA PGKSGSETPGTSESATPESGGGEVOLVESG
CACCCGAAAGTGGIGGCGGAGAGGTTCAG
CTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTIGTCCT
GGLVQPGGSLRLSCAASGFNISYSSIHWVR
GIGCAGCTTCIGGCITCAACATCTCTTATTCTICTATCCACTGGGIGCGTCAGGCCCCGGGTAAGGGCCIGGAATGGET
TGC.ATAT QAPG KG L E
VYVATISSYYGYTYYADSVKG RF
ATTTMCITATTATGGCTATACITATTATGCCGATAGCGICAAGGGCCGTTICACTATAAGCGCAGACACATCCAAAAAC
ACAGCC TISADISKNTAYLQMNSLRAEDTAVYTC.AR

IGCTCGCGCTCATTACITCCCGTGGECTGGTGCTAT
AHTFPWAGAMDYWGQGTLVTVSSGEGG
GGACTACTGGGGICAAGGAACCCTGGTCACCGTCTCCTCGGGIG
GAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCC
SDIQNITQSPSSLSASVGDRVTITCRASQSV
CTGICCGCCICIGTGGGCGATAGGETCACCATCACCTGCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAGCCIGGTATC
AACAGAA SSAVAWYQQKPGKAPKLLIYSASSLYSGVP

TTCCGG SRFSGSRSGIDFTLTISSIQPEOFATYYCQQ

ATCACGT YYWPITFG QGTKV El K

TCGGACAGGGTACCAAGGTGGAGATCAAA
No C
0) I-a Ln N) N) TABLE lA

ID DNA
SEQ Protein SEQ.
ID

ID 5 it GAGGTTCAGCTGGTGGAGTCTGGCGGIGGCCTGGTGLAGCCAGGGGGCTCACTCCGITTGTCCTGTGCAGCTTCTGGCT
TCAACA 19 EVQLVESG GGI.VQPGGSLRLSCAASGFNIS 20 hole-ICTCTTATTATTATATGCACTGGGIGCGTCAGGCCCCEGGTAAGGGCCTGGAATGGGTTGCATCTATTICTTCTTATTA
TGGCTCTA YYYMHWVROAPGKGLEINVASISSYYGiT

CTTATTATGCCGATAGCGTCAAGGGCCGTTICACTATAAGCGCAGACACATCCAAAAACACAGCCTAcCTACAAATGAA
CAGCTTA YYADSVKGRFTISADTSKNTAYLQMNSLRA

AGAGCTGACGACACTGCCGTCTATTATTGTGCTCGCTCTTGGTGGGCrrGGGcrrrrGACTAcTGsGGTCAAGGAACCC
TGGTCAC EDTAVYYCARSWWAWAFDYWGQGTLVT
CGTCTCCTCGGGTGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGICCGCCTCTGIGGGCGATAGG
GICACC VSSGGGGSDIO.mTOSPSSLSAsvGDRVTIT
ATCACCTGCCGTGCCAGTCAGTCCGTGICCACCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTC
TGATTTA CRASQSVSSAVAWYQQKFGKAPKWYSAS
CTCGGCATCCAGCCTCTACTCTGGAGrcccrrcrcGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATC
AGCAGTCT
SLYSEVPSRFSGSRSGTDFTLTISSLQPEDFA
GCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTACTCTGTTTACGCTICTCTGATCACGTTCGGACAGGGT
ACCAAGG TYYCQQHYSVYASLITFGQGTKVEIKLEDKT
TGGAGATCAAACTCGAGeaceaaattcacacaAAAGTIGAGCCCMATCTTCTgateagacccateetTGCCCACCGTGC
CCAGCACCTGAA HTKVEPKSSDKTHNCPPCPAPELLGGPSVF
CTCCTGGGGGGACCGTCAGTMCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACA

GGIGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACIGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG
ACAAA EVKFNWYVDGVEVHNAKTKPREEQYNST
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGIGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGA YRWSVOVLHODWLNGKEYKCKVSNKAL
GTAC.AAGTGCAAGGICTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG
AGAACCA PAFIEKTISKAKGQPREPQVYTLPPIRELMT
CAGGTGTACACCCIGCCCCCAATCCGGGAGCTGATEACCAGCAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCT
ATCCCA SNQVSLSCAVKGFYPSDIAVEWESNGQPE
GCGACATCGCCGTGGAGTGGGAGAGCAATEGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT

CCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTICTCATGCTCCGTGATGCATGAGGC
QQGNVFSCSVMHEALHNHYMKSISLSPG
TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAG
TCCGCC KSGSETPGTSESATPESGGGEVQLVESGGG
ACACCCGAAAGTGGIGGCGGAGAGGTICAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTT

TGTGCAGCTTCTGGCTTCAACATCTCTTCTTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGG
TTECATC PGKGLEWVASIYSSYGYTSYADSVKGRFTIS
TATTTATTCTICTTATGGCTATACTTCTTATGCCGATAGCGTCAAGGGCCGTTICACTATAAGCGCAGACACATCCAAA
AACACAGC ADTSKNTAYLQMNSLRAEDTAVYYCARTV
CTACCTACAAATGAACAGCTTAAGAGCTGAG
GACACTGCCGTCTATTATTGTGCMGCACTGTTCGTGGATCCAAAAAACCETACT
RGSKKPYFSGWAMDYWGQGTLVTVSSG
TCTCTGGITGGGCTATGGACTACTGGGGTCAAGGAACCCTGGICACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCA
GATGAC GGGSDIQMTQSPSSLSASVGDRVTITCRAS
CCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGICACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGC
GCTGTAG QSVSSAVAWYQQKPGKAPKWYSASSLYS
CCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGMCTGATTTACTCGGCATCCAGCCrCTACTCTGGAGTCCCTTCTC
GCTTCT GVPSRFSGSRSGTDFTLTISSLQPEDFATYY
CTGGTAGccErfCCGGGACGGATTICACTCTGACCATCAGCAGTCTSCAGCCGGAAGACTTCGCAACTTATTACTGICA
GCAATAC CQQYSWGPFTFGQGTKVEIK

TCTIGGGETCCGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA

WC
0"(j1 N) NJ
TABLE IA
ID DNA
SEQ Protein SEQ k.g ID

ID

CAGCCAGGGGGCTCACTCCGTUGICCIGTGCAGCTICTGGCTICAACat 21 EVQLVESGGG

knobs ctcttattattatatcCACTGGGTGCGTCAGGCCCOGGGTAAGGGCCTGGAATGGGTTGCATCTATTTATTCTTCITCT

TATGCCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCT
TAAGAGC ADSVKGRFTISADTSKNTAYLQM NSLRAE D
2542 TGAGGAC.ACTGCCGTCTATTATTGTG [ICC CICTICTTACGCTTG
GGCTATMACTACIGGGGTCAAGGAACCCTGGTCACCGTCIC TAVYYCARSSYAWAI DYWGQGTLVTVSSG
CTCGGGTGGAGGTGGCAGTGATATCCAGATGACCC.AGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCAC
CATCACC GGGSDIQM TaSPSSLSASVG DRVTITCRAS
TGCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCIGATTT
ACICGGC QSVSSAVAWYQQKPGKAPK LLITSASSLYS
ATCCAGCCICTACTCTGGAGTCCCTTCTCGCTTCYCIGGTAGCCGTTCCGGGACGGAMCACTCTGACCATCAGCAGICT
GCAGCC GVPSRFSGSRSGTDFTLTISSLQP EDFATYY
GGAAGACTTCGCAACTTATTACTGTCAGCAATCTEGTIGGTEGGGTGTTTCTCTGATCACGTTCGGACAGGGTACCAAG
GTGGAG CQQSGWWGVSLITFGQGTKVEIKLEDKTH
ATCAAA CTCGA Gga cases ctca ca caAAAG TG GAG CCCAAAACTICTgata agaccca tactTG C
CCA CCGTG cam CACCTGAACTC CT TKVEP KTSDKTHTCPPCP APE LLGG PSVF LP
GGGGGGACCGTCAGICTICCTCTICCCCCC.AAAACCCAAGGACACCCTCATGATCTCCCOGACCCCTGAGGTCACATG
CGTGGTG G PPKPKDTL M ISRTPE VTCVVVDVSH ED PEV
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAcGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGC KFNWYVDGVEVH NAKTKPR EEQYN STYR
GCGAGGAG
CAGTACAACAGCACGTACCGTGTEGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
VVSVLTVLHQOWLNGKEYKCKVSNKALPA
AGTGCAAGGTLICCAACMAGCCCTCCCAGCCOCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
ATGET PIEKTJSKAKGP REPMVFOLPPSREEMTK
GITTGACCIGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGIGGTGCATGETCAAGGGCTTCTATCCC
ACCGAC N QVSLWCIVIVKGFYPSDIAVEWESNGQP E
ATCG
CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCUTGCTGGACTCCGACGGCTCCIT
CT N NYKTIPPVLDSDGSFFLYSKLIVEIKSRWQ
TCCIGTACAGCAAGCTCACCGTGGACAAGAGCCGCTGGCAGCAGGGGAACGICTICTCATGCTCCGTGATGCATGAGGC

CAACCACTACACGCAGAAGAGCCTCTCCCTGICTCCGG
GTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCECCACACCC SGSETPGTSESATPESGGG
EVQLVESGGGL
GAAAGTGGIGGCGGAGAGGTTCAGCIGGIGGAGICTGGCGGTGGCCTGGTGCAGCCAG
GGGGCTCACTCCGTTIGTCCTGTGCA VQPGGSLRLSCAASGFNISYSSIHWVRQAP
GCTICTGEITCAACATCTCTIATTCTTCTATCCACTGEGTGCGTCAGGCCCCGGGTAAG
GGCCIGGAATGGGITGCATATATTICT G KGLEW VAYISSYYGYTYYADSVKG RFT' SA
TCTTATTATGGCTATACTIATTATGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAG
CCTACCTA DTSKNTAYLQMNSLRAEDTAVYYCARANY
CAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTICCCGTGGGCTG
GTGCTATGGACTA FPWAGAMDYWG QGT LVTVSSGG GGSDI
CTGGGGICAAGGAACCCTGGICACCGTCTCCTCGGGIG
GAGGIGGCAGTGATATCCAGATEACCCAGTCCCCGAGCTCCCTGICC
OJVITQSPSSLSASVGDRVIITCRASQSVSSA
GCCICTGTGGGCGATAGGGICACCATCACCTG CCGTG
CCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAG VAWYQQKPGKAPK
LLIYSASSLYSGVPSFIF
GAAAAGCTCCGAAGCTICTGATTTACTCGGCATCCAGCCTCTACICTEGAGTCCCTTCTCGCTTCTCTGGTAG
CCGTTCCGGGACGG SGSRSGTDFILTISSLOPEDFATYYCQQYY
ATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTIATI"ACTGTCAGCAATACTACTGGCCGAICA

AGGGTACCAAGGTGGAGATCAAA
k=de tn C
0) I-a Ln N) N) No ID DNA
SEC( Protein SEC!
ID

I D 5 it CaGGIGCAGCCAGGGGGCTCACTCCGTITGICCTGTGCAGCTTCTGGCTTCAACet 23 hole-ctcttattattatateCACTGGGTGCGTCAGGCCCCOGGTAAGGGCCIGGAATGGGTTGCATCTATTTATTCTICTICT
AGCTATACTTAT YYYu1WVRcAPGKcLEWVAs1YssssYrYY

CCTACCIACAAATGAACAGCTTAAGAGC ADSVKGRFTISADTSK
NTAYLQM NSLRAED

TGAGGACACTECCGTCTATTATTGTGCTCGCTCTTCTTACGCTIGGGCTATTGACTACTGGGGICAAGGAACCCTGGIC
ACCGTCTC TAWYCARSSYAWAIDYWCOGTLVIVSSG
CTCGGG TGGAGG TGG CAE TGATATCCAGATGACCCAGTCCCCGAG CTCC CTGTCCG CCTCTG TG G G
CGATAGGGTCACCATCACC GGGSDIQMTQWSSLSASVGDRVTITCRAS
TGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTT
ACTCGGC QSVSSAVAWYQQKPGKAPKWYSASSLYS
ATCCAGCCICTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTICCGGGACG
GATTTCACTCTGACCATCAGCAGTCTGCAG CC
GVPSRFSGSRSGTDFTLTISSLQPED FATTY
GGAAGACTTCGCAACTTATTACTGTCAGCAATCTGGTTG GTGG
GGTGTTICTCTGATCACGTTCGGACAGGGTACCAAGGTGGAG

ATCAAACTCGAGgacaa aactcacaceAAAGTTGAGCCCAAATCTTCTgataagaccutaatTGCCCACCGTGCCCAGCACCTGAACTCCT
TKVEPKSSDKTHNCPPCPAPELLGGPSVFLE

CGTGGIGG PP KPK DT LM ISRTP E VT DNA, TGGACGTGAGCCACGAAGACCCTGAGGICAAGTTCAACTGETACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGC KFNWYVDGVEV NAKTKPREEDYNSTYR
GGGAGGAGCAGTACAACAGCACGTACCGTGIGGICAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACA VVSVLTVLHODWLN GKEYKCKVSNKALPA
AGTG
CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG

GTACACCCTGCCCCCAATCCGGGAGCTGATGACCAG CAACCAGGTCAG
CCrGAGCTGCGCCGTCAAAGGCTTCTATCCCAG CGAC
QVSLSCAVKGFYPSD IAVEWESNGQPEN N
ATCG CCG TGG AGTGGGA GAG CAATG GGCAG CCGG AG AACAACTACAAG AC CACGCCTC CCGTG
CTGGACTCCGACGG CrCCUCT YKTTPPVLOSDGSFFLVSKLTVD
KSRWOO., TCCTCGTGAGCAAGCTCACCGTGGACAAGAG CAGGTGG
CAGCAGGGGAACGTCTICTCATGCTCCGTGATGCATGAGGCTCTG CA

CAACCACTACACGCAGAAGAGCCICTCCCTGTCTCCGGETAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCC

GGEVQLVESGGGLV

CCAGGEGGCTCACTCCGTTTGTCCTGTG CA

GCTTCTGGCTTCAACATCTCTICTTATTATATCCACTGGGTGCGTCAGGCCCCOGGTAAGGGCCTGGAATGGGTIGCAT

TCTICTTATGGCTATACTTCTTATG

TSKNTAYLQMNSLRAEDTAVYYCARIVRG
CAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACT
ICTCTGG SICK PYFSGWAMDYWGQGTLVIVSSGGS
TTGGGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACC
CAGTCC GSDIQMTQSPSSLSASVGDRVTITCRASQS
CCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACC.ATCACCTGCCGTGCCAGICAGTCCGTGTCCAGCGCTGTA
GCCTGGTA VSSAVAINYQQKPGKAPK LUYSASSLYSGV
TCAACAGAAACCAG GAAAAGCTCCGAAG CTTCTGATTTACTCGGCATCCAG
CCICTACTCTGGAGTCCCTICTCGCTTCTCTGGTAG PSR
FSGSRSGTDPTLTISSLQPEDFATYYCQ
CCGTTCCGGGACGGATTTCACTCTGACCATC.AG
CAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTCTTGGG

GTCCGTTCACGITCGGACAGGGTACCAAGGTGGAGATCAAA
No S-D
fro C
0) I-a N) NJ
I-a co.
No ID DNA
SEQ Protein SE Q
I D

knob- TCTCTTATTATTATATCCACTGGGTGCGTCAGGCCCCGEGTAAGGGCCTGGAATGGGITG
CATCTATTTATCCTTCTICTGGCTATA Y'YYI HWVROAPGKGLEWVASlYPSSGYTIN

CGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
ADSVKGRFTISADTSKNTAYLQMNSLRAED

GGTCAC TAVYYCARSSFYWAMDYWGQGTLVTVSS

G GTCACC GGGGSDI QMTOSPSSLSASVGDRVIITC RA
ATCACCTGCCGTGCCAGTC.AGICCGTGTCCAGCGCTGTAGCCIGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTI
CTGAUTA SQSVSSAVAWYQQKPGKAPKLLIYSASSLY
CTDGGCATCCAGCCTCTACTCTGGAGTCCCECTCGCTTCTCTG
GTAGCCGTTCCGGGACGGATITCAMTGACCATCAGCAGICT SGVPSRFSGSRSGTOFTLTISSLOPEDFATY
GCAGCCGGAAGACTTCGCAACTTATTACTGICAGCAATMACGCTGMACCTGTTCACGITCGGACAGGGIACCAAG
GTGGAGA YCQQSYAAYLFTFGOGTKVE IKLED KTHTK
TCAAACTCGAGsa ca a a actca cacaAAAGTGGAGCCCAAAACTICTgataagacccatactTG
CCCACCGTGCCCAGCACCTGAACTCCTG VEPKTSDKTHTCPPCPAPELLGGPSVFLFPP
GGGGGACCGTCAGICTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCG
TGGTEGT KPICCITLM ISRTPEVICVVVDVSH EDPEVKF
GGACGTGAGCCACGAAGACCCTGAGGICAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCG NWYVDGVEVHNAKTKPREEQYNSTYRVV
CGAGGAGCAGTACAACAGCACGTACCGTGIGGICAGCGTCCTCACCGICCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAA SVLTVLH QDWINGKEYKCKVSN KALPAPIE
GTGCAAGGTCTCCAACAAAG
CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAATG GIG KTISKAKG
QPR P MVF D LP PS RE E MTh NQ
TITGACCTG
CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTGETGCATGGICAAGGGCTICTATCCCAGCGACA
VSLWCMVKGFYPSDIAVEWESN GOREN N
TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
MCIT YKTIPPVIDSOGSFFLYSKLTVD KSRWQQG
CCIGTACAGCAAGCTCACCGTGGACAAGAGCCGCTGGCAGCAGGGGAACGICTICTCATGCTCCGTGATGCATGAGGCT
CTGCAC NVFSCSVMH EALHNHYTQKSLSLSPGKSG
AACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAAAGCGGCAGCGAGACTCCCG GGACCTCAGAGTC
CGCCACACCCG SET PGTSESATPESGGG EVOLVESG GGIVQ
AAAGTGGIGGCGGAGAGGTTCAGCTGGIGGAGTCTGG CGGTGGCCTGGTG
CAGCCAGGGGGCTCACTCCGTTTGICCTGIGCAG PG GSL R LSCAAS GFNISYSSIH WVR OAP G K
CITCTGGCTTCAACATCTCTTATTCTTCTATCCACTGGGIGCETCAGGCCCCG
GGTAAGGGCCIGGAATGGGTTGCATATATTTCTT G LEWVAY I SSYYGYTYYADSVKG RFT I S ADT
CTTATTATGCCTATACTTATTATGCCGATAGCGTCAAGG
GCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTAC
SKNTAYLQMNSLRAEDTAVYYCARAHYFP
AAATG AACAG CTTAAG AG CTGAG G ACACIGCCGTCTATTATTGIG CTCG CG CT CATTACTT C CUTE

TGEGGICAAGGAACCCIGGTCACCGTCTCCTCGGGTGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCC
IGTCCG MTO$PSSLSASVGDRVTITCRASQSVSSAV
CCTCTGTGGGCGATAGG
GICACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCIGTAGCCTEGTATCAACAGAAACCAGG
AWYQQKPGKAPKLUYSASSLYSGVPSRFS
AAAAGCTCCGAAGCTICTGATTTACTCGGCATCCAGCCICTACTCTGGAGTCCCTICTCGCTrCTCTGGTAGCCGTTCC
GGGACGGA GSRSGTDFILTISSLQPEDFATYYCQQYYW
TTTCACTCTGACCATCAG
CAGTCTGCAGCCGGAAGACTTCGCAACTTATTACT6TCAGC.AATACTACTGGCCGATCACGTTCGGACA

GGGTACCAAGGTGGAGATCAAA
No tit C
0) I-a Ln N) N) No ID DNA
SEQ Protein SEQ
ID

ID trot CAGCCAGGGGGCTC.ACTCCGTTTGTCCIGTGCAGCTICTGGCTICAACA 27 EVQLVESGG G LVQF GG S

hole-TCTCTTATTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATTTATCCTICITC
TGGCTATA YYYIHWVRQAPGKGLEWVASIYPSSGYTYY

CTIATTATGCCGATAGCGTCAAGGGCCGMCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACA
GCTTA ADSVKG RFTISADTSKNTAYLQMNSLRAED

GTCAAGGAACC CTG GTCAC TAVYYCARSSFYWAMDYWGQGTLVIVSS
CGTCTCCTCGGGTGGAG GIGGCAGTGATATCCAGATGACCCAGTCCCCGAG CTCCCTGTC CG CCTCTG TGGG
CGATAG G GTCACC GGGGSDICIMTQSPSSLSASVGDRVTITCRA
ATCACCTGCCGTGCCAGICAGTCCGTGTCCAGCGCTGTAGCCIGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTIC
TGATTTA SQSVSSAVAWYQQKPGKAPKLUYSASSLY
CTCGG
CATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAG
CAGTCT SGVPSRFSGSRSGTDFTLTISSLQPEDFATY

GTGGAGA YCQQSYAAYLFTFGQGTKVEIK LED KTHT K
TCAAACTCGAGga ca a a actcaoecaAAAGTTGAG CCCAAATCTICTgataa ga cccataatTGCCCACCGTGCCCAG CACCTGAACTCCTG VE P KSSDKTH N C P PC PAP E LLGG
PSVFLF P
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGIGGIGGT PKPKITTLMISRTPEVICVVVOVSHEDPEVK
GGACGTGAGCCACGAAGACCCTGAGGTCMGTICAACTG
GTACGTGGACGECGTGGAGGTGCATAATGCCAAGACAAAGCCGCG FNWYVDG VEVHNAKTKPREEQYNSTYRV
GGAGGAGCAGTACAACAGC.ACGTACCGTUTG
GICAGCGTCCICACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA VSVLTVL
HQDWINGKEYKCKVSN KALPAP
GTGCAAGETCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGTG I EKTISKAKG QPR EP QVYTLP PI R ELMTSN Q
TACACCCTGCCCCCAATCCGGGAGCTGATGACCAGCAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCC.

CGCCGTGGAGTEGGAGAGCAATGGECAGCCGGAGAAC.AACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCITCTIC KTTPPVLDSOGSFFLVSKLTVDKSRWQQG
CTCGTG AGCAAG CTCA C CGTG GA CAAGAG CAGGTG G CAG C.A6 GG G AAC GTCTTCTCATG CT
CCGTGATGCATG AGG CTCTGCACA NVFSCSVM HEA LH N HYTQKS LS LSPG KSG
ACCACTACACG CAGAAGAGCCTCTCCCIGTCTCCGGGIAAAAG
CGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCCGA SETPGISESATPESGGGEVOLVESGGGLVQ
AAGTGGTGGCGGAGAGGTTCAGCFGEIGGAGICTGECGGIGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGICCTGT
GCAGC PGGSLRLSCAASGFNISSYYINWVROAPG1( TTCTGGCTICAACATCTCTICTTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAAIGGG'TTGCATC
TATTTATTC GLEWVASIYSSYGYTWADSVKGRFTISADT
TTCTTATGGCTATACTTMATGCCGATAG0GTCAAGGGCCGTTTC.ACTATAAGCGCAGACACATCCAAAAACACAGCCT
ACCTACA SKNTAYLQMNSLRAEDTAVYYCARTVRGS
AATGAACAGCTTAAGAG
CTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACTTCTCTG GTT
KKPYFSGWAMDYWGQGTLVTVSSCGG G
GGGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTG

GAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGICAGTCCGTGTCCAGCGCTGTAGCC
TGGTATC SSAVAWYQQKPGKAPKLUYSASSLYSGVP
AACAGAAACCAGGAAAAGCTCCGAAGCITCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTC
TGGTAGCC SRFSGSRSGTOFTLTISSLCWEDFATYYCQQ
GTTCCGGGACGGATTICACTCTGACCATCAGCAGICIG
CAGCCGGAAGAMCGcAAmATIACTGICAGCAATACTCTIGGGGT YSWGPFTFGO,GTKVEIK 1-3 CCGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA
No S.D.
en C
0) I-a Ln N) N) TAB LE lA

No ID DNA
SE Q Protein SEQ
ID

ID

knob-TCTCTTATTATTCTATGCACTGEGTGCGTCAGGCCCCGGGTAAGGGCCIGGAATGGGTTGCATCTATTICTICTTATTA
TAGCTCTA YYSPAHWVRQAPGKGLEWVASISSYYSSTS

CCGATAGCGTCAAGGGCCGMCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGaTA
YADSVKGRFTISADTSKNTAYLO,MNSLRAE

GICAAGGAACCCTEGTCACCG DTAVYYCARFWYPGMDYWGQGTLVTVSS
ICTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTcCaGAGCTCCCIGTCCGCCTCTGTEGGCGATAGGGTC
ACCAT G GGGSDIQMTQSPSSLSASVG DRVTITCRA
CACCTG CCGTGCCAGTCAGICCGTGTCCAGCGCTGTAG CaGGTATCAACAGAAACCAGGAAAAG
CTCCGAAGCTTCTGATTTACT SQSVSSAVAWYQQKPGKAP KLUYSASSLY
CGGCATCCAGCCICTACTCTGGAGICCCTICTCGCTICTCTEGTAGCCGITCCGGGACGGATTTCACTCTGACCATCMC
AGTCTG C SGVPSRFSG SIISGTDFTLTISS LQPEDFATY
AGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAC.ATIGGICTTACCCGATCACGTTCGEACAGGETACCAAGGTGG
AGATCAA YCQQHWSYPITFGQGTKV El KLEDKTHTKV
ACTCGAGga ca a a a eta co caAAAGTGGAGCCCAAAACTICTgata agaceca ta cflt CCCACCGTGCCCAG CACCTGAACTCCTGGGG
EPKTSDKTHTCPPCPAPELLGGPSVFLF PPK
GGACCETCAGTCTICCTCTICCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGTEGA PKDTLMISRTPEVTCVVVDVSHEDPEVK FN
CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGCGA WYVDGVEVH NAKTKPREEQYNSTYRVVS

AAGTGC VLTVLHQDWLN G KEYKCKVSN K ALP API E
AAGGICICCAACAAAGCCCICCMCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCAATGGIGMG
KTISKAKGQPREPMVFDLPPSREE MTK NQ
ACCTGCCCCCATCCCGG
GAGGAGATGACCAAGAACCAGGTCAGCCMTGGTGCATEGTCAAGGGCTTCIATCCCAGCGACATCG C
VSLWCMV KG FYPSDIAVEWESN GQPENN
CGTGGAGTGGGAGAG CAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACG
GCTCCTTCITCCTG YKTIPPVLDSDGSFFLYSKLTVDKSRWQQG
TACAG CAAG CT CACCG TG GACAAG AG CCGCMG CAGCAG GG GAAC ISTCTTCTCATG CTCCGTGATG
CATGAG G CT CNCACAACC N VFSCSVMHEALH NHYTCtIGULSPG KSG
ACIACACGCAGAAGAGCCICTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACC
CGAAAG SETPGTSESATPESGGGEVQLV ESGGE LVQ

GGCTTCAACATCTCTTATICTTCTATCCACTGGGIGCGTCAGGCCCCGGGTAAGGGCCIGGAATGGGTIG
CATATATTICTICTTAT G LEWVAYISSYYGMYADSVKGRFTISADT
TATGG CTATACITATTATGCCGATAGCGTCAAGGECCGMCACTATAAGCGCAGACACATCCAAAAACACAG
CCTACCTACAAAT SKNTAYLQMNSLRAEDTAVYYCARAHYFP
GAACAGCTIAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTICCCGTGGGCTGGTGCTAIGGAC
TACTGG G WAGAM DYWGQGTLVIVSSGGGGSD IQ
GICAAGGAACCCTGGTC.ACCGICTCCTCGGGIGGAGGIGGCAGTGATATCCAGAIGACCCAGTCCCCGAGCTCCCIGT
CCGCCTCT MTOSPSSLSASVGDRVTITCRASQSVSSAV
GIGGECGATAGGETCACCATCACCTOCCGTGCCAGICAGTCCGTGICCAG
CGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAG AWYQQKPGKAPKLINSASSLYSGVPSRFS
CTCCGAAGCTTCTEATTTACTCG GCATCCAGCCICTACTCTGGAGTCCCTTCTCGCTTCTCTGGIAG
CCGTTCCGGGACGGATTICA GSRSGTDFTLTISSLQPEDFATYYCQQYYW
=
CICTGACCATCAGC.AGTCTGCAGCCG
GAAGACTTCG CAACTTATTACTGTCAGCAATACTACTGGCCGAICACGITCG GACAGGGT P ITFGQGTKVE

ACCAAGGTGGAGATCAAA
No en C
0) I-a Ln N) N) I-a ID DNA
SEQ Protein SEQ
ID

ID rnt ¨GAGGTTCAGCTGGTGGAGTCMGCGGIGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTIGTCCIGTGCAGCTTCTGGCT
TCAACA 31 EvQLVESGGGLVQPGGSLRLSCAASGFNIS 32 hole-TCTCTTATTATTCTATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGITGCATCTATTTCTTCTTATTA
TAGCTCTA YYSMHWVROAPGKGLEWVASISSYYSSTS

CTICTTATGCCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAMCACAGCCIACCTACAAATGAAC
AGMA YADSVKGRFTISADTSKNTAYLRIVINSLRAE

AGAGCTGAGGACACTGCCGICTATTATTGTGCTCGCTTCTGGTACCCGGETATGGACTACTGGGGICAAGGAACCCIGG
ICACCG DTAVYYCARPWYPGMDYWGQGTLVTVSS
TCTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCIGTCCGCCICTGTGGGCGATAGGGT
CACCAT GGGGSDIQMTQSPSSILSASVGDRVTITCRA
CACCTGCCGTGCCAGICAGTCCGTGTCCAGCGCTGTAGCCTEGTATCAACAGAAACCAGGAAAAGCTCCGMGCTICTGA
TTTACT SQSVSSAVAWYQQKPGKAPKWYSASSLY
CEGCATCCAGCCICTACTCTGGAGTCCCITCTCGCTICTCTGGTAGCCGTTCCGGGACGGATTTCACTCTEACCATCAG
CAGTCTGC SGVPSRFSGSRSGTDFTLTISSLQPEDFATY
AGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACATTGGICTTACCCGATCACGTTCGGACAGGGTACCAAGGTGGA
GATCAA YCQQHWSYPITFGQGTKVEIKLEDKTHTKV
ACTCGAGga caa aactcaca caAAAGTIGAGCCCAAATCTTCTgata agacccataatTGCCCACCGTGCCCAGCACCTGAACTCMGGGG
EPKSSDKTHNCPPCPAPELLGGPSVFLFPPK
GACCGICAGICITCCTMCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG
IGGAC PKDILMISRTPEVTCVVVDVSHEDPEVKFN
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGIGCATAATGCCAAGACAAAGCCGC

GAGCAGTACAACAGCACGTACCGTGTGGICAGCGTCCTCACCGTCCTGCACCAGGACrGGCTGAATGGCAAGGAGTACA
AGTGCA VLIVLHODWINGKEYKCKVSNKALPAPIE
AGGTCTCCAACAAAGCCCTCCCAGCCCCC.ATCGAGAAMCCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACAC KTISKAKGOPREPQVYTLPPIRELMTSNQV
CCTGCCCCCAATCCGGGAGCTGATGACCAGCAACCAGGICAGCCTGAGCTGCGCCGTCAAAGGCTICTATCCCAGCGAC
ATCGCC SLSCAVKGFYPSDIAVEWESNGQPENNYK
GIGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCITCT
TCCTCG TTPPvLDsDG5FFLvSKLTVDKSRwQQGN

nicrÃCACAACCA
VFSC.SVMHEALHNHYTO.KSLSLSPGKSGSE
CTACACGCAGAAGAGCCTCTCCCTGICTCCGGGTAAMGCGGCAGCGAGACIt CCUGGACCICAGAGTCCGCCACACCCGAAAGT
TPGTSESATPESGGGEVQLVESGGGLVQP

CTTCT GGSLRLSCAASGFNISSYYIHWVRQAPGKG

ATTCTTCT LEWVASYSSYGYTRADSVKGRFTISADTS
TATGGCTATACTTCTTATGCCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAAACACAGCCTACC

GAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTAITGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACTFCTCT
GGTTGGG KPYFSGWAMDYWGQGTLVTVSSGGGGS
CTATGGACTACTGGGGICAAGGAACCCTGGICACCGTCTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCATIC
CCCGAG DIQMTQSPSSLSASVGDRVTITCRASQSVS
CTCCCTGICCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGG
TATCAAC SAVAWYQQKPGKAPKLUYSASSLYSGVPS
AGAAACCAGGAAAAGCTCCGAAGCTTCTGArTTACTCGGCATCCAGCCTCTACICTGGAGTCCCTICTCGCTICTCTGG
TAGCCGITT RFSGSRSGTDFILTISSLCIPEDFATYYCQQY
CCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGICAGCAATACTOTGG
GGICCG SWGPFTFGQGTKVEIK

TICACGTICGGACAGGGTACCAAGGIGGAGATCAAA
en C
0) I-a Ln N) N) TABLE lA

ID ONA
SEQ Protein SEQ
I D

ID 5 it GAGGTTCAGCTGGIGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTIGICCTGTGCAGCTICTGGCT
TCAACC 33 EVO.LVESGGGLVQPGGSLRLSCAASGFN LS 34 knob- TCTCTTATTATTATATG CA CTGGGTG CG TCAGGCCCCGG GTAAGG GCCTGGAATG
GGTTGCATCTATTTATTCTTATTCTG GCTATA
YYYMHWVRQAPGKGLEWVASI YSYSGYT

CTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAA
CAGCTTA YYADSVKG RFTISADTSKNTAYLQMNSLRA
2542 AGAGCTGAGGAC.ACTGCCGTCTATTATTGTGCTCGCTCTTCTTTCGCTTG
GGCTTTTGACTACTGGGGTCAAGGAACCCIGGICACC
EDTAVYYCARSSFAWAFDYWGQGTLVTV
GICTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGIGGGCGATAGGG

TCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTG
GTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTAC
RASQSVSSAVAWYQQKPG KAPKWYSASS
TCGGCATCC.AGCCTCTACTCTGGAGICCCTTCTCGCTICTCTGGTAGCOGTTCCGGGACGGATTTCACTCTGACCATC
AG CAGTaG
LYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT
CAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGIGGTTGGGGTCCGUCACGTTCGGACAGGGTACCAAGGIGGA
GATCA YYCQQGGWG PFTFGQGTKVEI K LED
KTHT
AACTCGAGgacaaaactca taceAAAGTGGAGCCCAAAACTICTgataagacecatactTGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
KVEPKTSDKTHTCPPCPAPELLGGPSVFLFP
GGACCGTCAGICTICCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG
TGGIGGA PKP KDTLMISRTPEVTCVVVDVSH EDP
EVK
CGTGAG CCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTG CATAATG
CCAAGACAAAGCCGCGCGA FNWYVDGVEVHN A KT KP
R E EQYNSTYRV
-41 GGAGC.AGTACAACAGCACGTACCGTGTGETCAGCGTCaCACCGTCCIG
CACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
VSVLTVLHQDWLNGKEYKCKVSNKALPAP
oo AAGGICTCCAACAAAG C CCTCCCAGCC CCCATCG AGAAAACCAT CT C
CAAAG CCAAAGGG CAG C C CCG AG AACCAATG GTGTTTG I EKTISKAXGREPMVFDLF
PSREEMTKN
ACCTG CCCC CATCCCG GGAGGAG ATGACCAAG AA C CAGG TCAG CCTGTG GTG CATGGTCAAG GG
CTTCTATCCCAG C GACATCG C QVSLWCMVKGFYPS DI AVEWESN G
(WEN
CGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CT GACTCCGACGG
CTCCTTCTT CCTG
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
TACAGCAAGCTCACCGTGGACAAGAGCCGCTGGCAGCAGGGGAACGICTICTCATECTCCGTGATG CATE AGG CT
CTG CACAACC GNVFSCSV MH EA LH N
HYTOKSISLSPGKS
ACTACACECAGAAGAGCCICTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACC
CGAAAG GSETPGTS ESATP E SG G G EVCILV
E SGGE LV
TGGTGGCGGAGAGGITCAGCTGGIGGAGTCTGGCGGTGGCCIGGTGCAG CCAGGEGGCTCACTCCGTTIGTCCTGIG
CAGCTTCT QPGGSLRLSCAASG FNISYSSI
HVVVRQAPG
GGCTICAACATC1C7TATTCTICTATCCACTGGGIGCGTCAGGCCCCGGGTAAGGGcaGGAATGGGITGCATATATTTC
TTCTTAT KGL E WVAY I SSYYGYTYYADSV
KGRFTISAO
TATGG CTATACTTATTATGCCGATAGCGTCAAGGECCGTTICACTATAAGCG CAGACACATCCAMAACACAG
CCTACCTACAAAT
TSKNTAYLOMNSLRAEDTAVYYCARAHYF
GAACAGCTTAAGAGCTGASGACACTGCCGICTATTATTGTECTCGCGCTCATTACTTCCCGTGGGCTGGIGCTATEGAC
TACTGG G PWAGAMDYWGQGTLVTVSSGGGGSDI Q
GTCAAGGAACCCTGGICACCGICTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTC
CGCCICT MTQSPSSLSASVGDRVTITCRASQSVSSAV
GTGGGCGATAGGETCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAG

AWYQQKPGKAPKLLI YSASSLYSGVPSRFS
CTCCGAAGCTTCTGAMACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAG
CCGTTCCGGGACGGATTTCA
GSRSGTDFILTISSLQPEO FATNYCQQYYW
aCTGACCATCAGCAGTCTGCAGCCGGAAGACTICGCAACTTATTACTGTCAGCAATACTACTGG CCGATCACGTTCG
GACAGG GT PTTFGQGTKVEIK

ACCAAGGTGGAGATCAAA

C
0) I-a Ln N) N) No ID DNA
SEQ Protein SEQ
I D

ID 5 it c hole- TCTCTTATTATTATATGCACTGG GTGCGTCAGGCCCCGGGTAAGGGCCTGGAATG
GGITGCATCTATTTAITCTTATICTGGCTATA YYYMHWVROAPGKG
LEWVASIYSYSGYT

CGTC.AAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
YYADSVKGRFTISADTSKNIAYLQMNSLRA
2542 AGAG CTGAG GACACTG CC GICIATTATIGT G CTCGCTC'TTCTITCG CTTGGGCTT
TTGACTACTGGGGTCAAGGAACCCTGGICACC
EDTAVYYCARSSFAWAFDYWGQGTLVTV
GICTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCICTOTOGGCGATAGGG

TCACCTGCCGTGCCAGTCAGTCCGIGTCCAG C G CTGTAG CCM G TATCAACAGAAACCAG GAAAAG
CTCCGAAGMCTGATTIAC
RASQSVSSAVAWYQQKPGKAPKWYSASS
TCGECATCCAGCCTCTACTCTEGAGTCCCTICTCGCTICTCTGGTAGCCGTTCCGGGACG
GATITCACTCTGACCATCAG CAGTCTG
LYSGVP5RFSGSRSGTDFILTI Sal:IPED FAT
CAGCCG GAAGACTTCGCAACIIATTACIGTCAGCAAGGTGGTTGGGGICCGTTCACGTTCGGACAG
GGTACCAAGGTGGAGATC.A YYCQQGGWGPFTFGQGTKVE
I KLEDKTHT
AACTCG AGga caa a acted cacaAAAG TTGAG CCCAAATCTICTs at a agacccataatTGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
KVEPKSSDKTHNCPPCPAPELLGGPSVFLF

CGTGGIGGTGGA P PKP K DT L M
FSRTPEVTCVVVDVSH E DPE V
CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGA
KFNWYVDGVEVHNAKTKP REEQYNSTY R
GGAGCAGTACAACAGCACGTACCGTGTGGTMCGTCCICACCGTCCIGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGC VVSVLTVLHQDWLNGKEYKCKVSNKALPA
u:s A/4G TCTCCAACAAAG CCCTCCCA GC

EKTISKAKGQPREPQVYTLPP IR E LMTSN
CCCTGCCCCCAATCCGGGAGCTGATGACCAG
CAACC.AGGTCAGCCTGAGMCGCCGTCAAAGGCTICTATCCCAGCGACATCGCC
QVSLSCAV KG F'YPSD I AVEW ESNGQPEN N
GIG G AGTG G GAGAG CAATGG G CAG CC G GAGAACAACTACAAG ACCA CG CCTC CCG TG
CIGGACTC CGACG G CTCCTTCTTCCTCG
YKTTPPVLDSDGSFFivsKurvaKSRWQQ
TGAGCAAGCTCACCGTGGACAAGAGCAGGIGGCAGCAGGGGAACGTCITCTCATGCTCCGIGATCCATGAGGCTCTGCA
CAACCA G NVFSCSVMHEALHNHYTQKSISLSPGKS
CTACACCCAGAAGAGCCICICCCTGTCTCCGGGTAAAAGCGGCAG CGAG ACTCCCG GGACCTCAG AG TCCG
CCACACCCGAAAGT GSETPGISESATPESGGGEVQLVESGGG
LV
GGTG GCGGAGAGGITCAGCTGGTGGAGTCIGGCGGIGG
CCIGGTGCAGCCAGGGGGCTCACTCCGTTTGICCIGTGCAGCTICT
QPGGSLRLSCAASGFN ISSYYI H WVROAPG
GGCTTCAACATCTCTTCTTATTATATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATTT
ATTCTTCT KGLEWVASIYSSYGYTSYADSVKGRFTISAD
TATGGCTATACTICTTATG
CCGATAGCGTCAAGGGCCGMCACIATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAAT
TS( NTAYLQM NSLRAE DTAWYCARWRG
GAACAG
CITAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGITCGTGGATCCAAAAAACCGTACTICTCTGGTTGG
G SKKPYFSGWAMDYWGQGTIVTVSSGGG

CCGAG GSDIO,MTQSPSSLSASVGDRVTITCRASQS
CTCCCTGICCGCCTICTGIGGGCGATAGGGICACC.ATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGIAGCCT
G GTATCAAC VSSAVAWYQQKPGKAPKWYSASSLYSGV
AGAAACCAGGAAAAGCTCCGAAGCTICTGArrrACTCGGCATCCAGCCTCTACTCIGGAGTCCMCTCGCTICTCTGETA
GCCGTT PSRFSGSRSGTDFTLTISSLQPEDFATYYCQ
CCGGGACGGATTTCACTCTGACCATCAGcAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTCTTG
GGGTCCG CraWGPFTFGQGTKVEIK

TTCACGTTCGGACAGGGTACCAAGGIGGAGATCAAA
S.!

C
0) I-a Ln N) N) TABLE lA

No JO
DNA
SEQ Protein SEQ
ID

ID 5 it 2928. GAGGTTCACCIGGIGGAGTCTGGCGGIGGCCIGGTG

knob- TaCrTATTCTICTATCCACTGGGTGCGTCAGGCCCCG
GGTAAGGGCCIGGAATGGGTIGCATCTATTTATCCTICTTATAGCTCTA
YSSIHWVR OAPGKG LEWVASIYPSYSSTYY

CCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
ADSVKGRFTSADTSKNTAYLQMNSLRAE D
2542 AGAGCTGAG GACACTGCCGTCTATTATTGTGCTCGCTACTACG CTATGGACTACrGGG
GICAAGGAACCCTGGTCACCGICTCCIC
TAVYYCARYYAMDYWGQGTLVRISSGGG
G GGTG GAGGTG G CAGTGATATCCAGAIGACCCAGTCCCCGAGCTCCCTGICCGCCTCTGTGGGCGATAG
GGTCACCATCACCTGC GSDIQMTCLSPSSLSASVGD
RVTITCRASQS
CGTGCCAGTCAGTCCGTGTCGAGCGCTGTAGCCIGGIATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACT
CGGCATC VSSAVAWYQQKPGKAPKILI YSASSLYSGV
CAGCCTCTACTCIEGAGTCCCTTCTCGCTICTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTG
CAGCCGGA PSRFSGSRSGTDFTLTISSLO,P
EDFATYYCQ
AGACTTCGCAACTTATTACTGTCAG CAAGCMCIACTACCCGATCACGITCGGACAGGGTACCAAG
GIGGAGATCAAACTCGAGg QAFYYPITFGQGTKVEIKLEDKTHTKVE
aca3aactcacacaAMGTG6AGCCCAAAACrrcTgataagacccatactTGcccAccCrGcCcAGcAccTGAAcTccrG
GGG GGACCGTC SDKTHTCP PCPAPE
LLGGPSVFLFPPKPXDI
AGICTICCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC
LMISRTPEVTCWVDVSH ED PEV KFNWYV
ACGAAGACCCTGAGGICAAGTICAACTGGTACETGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGCGAGGA
GCAGT DGVEVH NAKTKPREEQYNSTYRVVSVLIV
oe ACAAC.AGCACGTACC.GTGIGGICAGCGTCCTCACCETCCIGCACCAGGACTGGCTGAATGGCAAG
GAGIACAAGIGCAAGGITTC LHQDWINGKEYKCKVSNKALPAPI EKTISK
o CAACAAAG CC CTCCCAG CCCC CATC G AGAAAACCATCTCCAAAG
CCAAAGGG CAG CCCCGAGAAC CAATGG TGTTTGACCTG C CCC
AKGQPREPMVFDLPPSREEMTKNQVSLW
CATCCCGGGAGGAGATGACCAAGAACCAGETCAGCCTGTGGTGCATGGTCAAGGGCTICIATCCCAGCGACATCGCCET

VIM?
GGGAGAG CAATGGG C.AG CCGGAGAACAACTACAAGACCACGCCTCCCGIGCTGGACTCCGACGG
CICCTTCTTCCIGTACAGCAA
PVLDSDGSFFLYSKINDKSRWQQGNWS
GCTCACCGIGGACAAGAGCCGCTGGC.AGCAGGGGAACGICTICTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACG CSVMHEALHNHYTQKSLSLSPOKSGSETP
CAGAAGAG
CCTCTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTC.AGAGTCCGCCACACCCGAAAGTEGTGGC
GTSESAII: ESGGGEVQLVESGGG LVQPGG
GGAGAGGITCAGCTGGIGGAGICTGGCGGIGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTIGTCCTGIGCAGCTICTG
ECTICA SLR LSCAASG FN
ISYSSIHWVRQAPGKGLE
A CATCTCTTATTCTICTATCCACIG GGTGCGTCAGGCCCCGGGTAAGGGCCTG GAATG GGTIG
CATATATTICTICITATTATGG CT
WVAYISSYYGYTYYADSVKGRFTISADTSK
ATACTTATTATGCCGATAGCGTCAAGGGCCGTITCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAAT
GAACAGC NTAYLQMN SLRAEDTAVYYCARAHYFPW
TTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTTCCCGTGGGCTGGTGCTATGGACTACTGGG
GICAAGG AGAM DYWGQGTLVTVSSGGGGSD I QMT
AACCCTGGTCACCGTCTCCTCGGGTG GAGGTG G
CAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGIGGGC

GATAGGGTCACCATCACCTGCCGTGCCAGTCAGICCGTGICCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAG
CTCCGA YQQKPGKAPK WYSASSLYSGVPSRFSGSR
AGCTICTGATTTACTCGGCATCCAG
CCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGA
SGTDFTLTISSLQPEDFATYYCQQYYWPITF
CCATCAGCAGTCTGCAG
CCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTACTGGcCGATCACGTTCGGACAGGGTACCAAG
G QGTKVEIK

GTGGAGATCAAA
No S-D

C
0) I-a Ln N) N) ID DNA
SEQ Prote In SEQ
ID

ID 5 it _ hole-TCTCTTATTCITCTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCIGGAATGGGTIGCATCTATTTATCCTICTTA
TAGCTCTA YSSIHWVROAPG KG LEWVASITPSYSSTYY

CTTATTATGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAA
CAGCTTA ADSVKGRFTISADTSKNTAYLQM NSLRAED

GTC.AAGGAACCCTGGTC.ACCGTCTCCTC

GGGTG GAG

G$01QMTQSPSSL,SASVGDRVTITCRASQS
CGTG
CCAGTCAGTCCGTGICCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGC
ATC VSSAVAWYQQK PG KAPKWYSA.SSLYSGV
CAGCCICTACTCTGGAGTCCCTICTCGCTICTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTG

AGACTTCGCAACTTATTACTGTCAG CAAGCTTICTACTACCCGATCACGTTCG
GACAGGGTACCAAGGTGGAGATCAAACTCGAGg QAFYYPITFG QGTKVE I KLEDKTHTKV E PKS
ca aa ctcaca ceAAAGITGAGCCCAAATCTICTgata aga =ate atTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA
SDKIHNCPPCPAPELLGGPSVFLFPPKPK D
GTCTTCCICTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAMCGTGGIGGTGGACGT
GAGCCA TLM ISRTP EVTCVVV DVS HE D PEV K
FN WY
CGAAGACCCTGAGGICAAGTTCAACTGETACGTGGACG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT ' VDGVEVHNAKTKPREEQYNSTYRVVSVLT

GCAAGGAGTACAAGTGCAAGGICTC VLFIQDWIN
GKEYKCKVSNKALPAPIEKTIS
CAACAAAGCCCTCCC.AGCCCCCATCGAGAAMCCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCC KAKGQPREPQVYTLPPIRELMTSNQVSLSC
CCAATCCGGGAGCTGATGACCAG CAAC C AG GTCAG CCTG AG CTG CG CCGTCAAAG G
CTTCTATCCCAGCGACATCG CCGTG GAST
AVKGFYPSDIAVEWESNGQPENNYKTTPF
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTICTTCCTCGT
GAGCAA VLDSDGSFFLVSKLTV DKSRWQQGN VFSC
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC.AIGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACG SVMHEALHNHYTQKSLSLSPGKSGSETPGT
CAGAAGAG CCTCTCCCTGICTCCG GGIAAAAG CG G CAG CG AGACTCCCG G GA CCTCAGAGTC CG
CCA CACCCGAAAGTG GIG GC SESATPESGGG
EVQLVESGGGLVQPGGSL
GGAGAGGTTCAGCTGGTGGAGICTGGCGGIG G CCT GGTG CAG C CAGG GG G CTCACTCC GMGTCCIGTG
CAGCTTCTG G CTT CA RLSCAASGFNISSYYIHWVRQAPGKGLEW

GGCT VASIYSSYGYTWADSVKGRFTISADTSK NT
ATACTTC'TIATGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAA
TGAACAGC AYLQMNSLRAEDTAVYYCARTVRGSKKPY
TTAAGAGCTGAGGACACTGCCGICTATTATTGTGCTCGCACTGITCGTGGATCCAAAAAACCGTACTICTCTGGITGGG
CTATGGA FSGWAM DYWGQGTLVWSSGGGGSDIQ
CTACTGGGGTCAAGGAACCCTGGICACCGICTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGC
TCCCTG NITQSPSSISASVGDRYTITCRASQSVSSAV
TCCG
CCTCIGIGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAGCCTGGTATCAACAGAA
ACC AWYQQKPG KAPKILI YSASSLYSGVPSRFS
AG GAAAAG CTCCGAAGCTTCTGATTTACTCGG CAT C CAGCCTCTA CTCTG
GAGTCCCTICTCGCTICTCIGGTAGCCGTTCCGGGAC
GSRSGTDFTLIISSLCIPEDFATYYCQQYSW
GGATTTCACICTGACCATCAGCAGTCTGCAGCCGGAAGACTI-CGCAACTTATTACTGICAGCAATACTCTTGGGGICCGTTCACGIT

CGGACAGGGTACCAAGGTGGAGATCAAA
en =

C
0) I-a Ln N) N) I-a No ID DNA
SEQ Protein SEQ
ID

I D

knob- TCGETICITCITCTATCCACTGGGTGCGTCAGGCCCCGG
GTAAGGGCCTGGAATGGGTTGCATCTATTTATTCTGCTITTGCCICTA
SSSEEPAIVR QAPGKGLEWVASIYSAFASTSY

CAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA

AGAGCTGAGGAC.ACIGCCGICTA17ATTGTGCTCGCTACCATITCCCGTTCGGITTTGCTITGGACTACIGGGGICAA
GGAACCCT TAWYCARYFIFPFG FALDYINGOGILVIVS
GGICACCGTCICCTCG
GGIGGAGGIGGCAGTGATATCCAGATGACCCAETCCCCGAGCTCCCTETCCGCCICTGIEGG CGATAGG
SEGGESDI QiNATQSPSSLSASVGDRVIITCR
GICACCATCACCIGCCGTGCCAGTCAGTCCGTETCCAGCGCTGTAGCCTEETATCAACAGAAACCAGGAAAAGCTCCGA
AECTICT ASQSVSSAVAWIQQKPGKAPKILIYSASSL
GATTTACTCGGCATCCAG
CCTCTACTCIGGAGTCCCTTCTCGCTTCTCTGGTAECCGTTCCGGGACGGATTTCACTCTGACCATCAG
YSEVPSRFSESRSETDFTLTISSLQPEDFAT
CAGTCIGCAGCCGGAAGACTICGCAACTTATTACTGICAGCAAGGTETTTACCTETTCACGTTCEGACAGEGTACCAAG
GTGGAG YYCQQGVYLFTFGQGTKVE IKL EDKTHTKV
ATCAAACTCGAGgataaaactica cacaAAAGIGGAGCCCAAAACITCTgataagacccatactTG
CCCACCGTGCCCAGCACCTGAACTCCT EPKTSDKTHTCP
PCPAPELLGGF'SVFLF PP K
GGGGEGACCGTCAGICTTCCTCITCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGEACCCCTGAGETCACATEC
GTGGTG G PKDTLM I SRTPEVTCVVVDVSH
EDPEVKFN
IGGACGTGAGCCACGAAGACCCTGAGETCAAETTCAACTGETACEIGGACGGCGTGEAGGTGCATAATGCCAAGACAAA
GCCGC WYVDGVEVHNAKTKPREEQYNSTYRWS
oe GCGAGGAGCAGIACAACAGCACGTACCGTGIGGTCAGCGTCCICACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA
GTACA VLTVLHQDWLNGKEYKCKVS NKALPAPI E
AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG
GGCAGCCCCGAGAACCAATGGT
KTISKAKGQPREPMVFDLPPSR EENITKNQ
GTTTGACCTG
CCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGTEGTGCATGGICAAGGGCTTCTATCCCAGCGAC
VSLWCMVKGFYPSDAVEWESNGQPENN
ATCG
CCGTGGAGTGGGAGAGCAATGEGCAGCCGGAGAACAACTACAAGACCACECCTCCCGTGCTGGACTCCEACEGCTCCTI

TCCTGTACAGCAAGCTCACCGTGGACAAGAGCCGCTGG CAGCAGGGGAACGTMCTCATGCTCCGTGATGCATGAG
GCTCTGCA NVFSCSVMHEALHNHYTQKSLSLSPGKSG
CAACCACTACACGCAGAAGAGCCICTCCCTGICTCCGGGTAAAAGCGECAGCGAGACTCCCGGGACCICAGAGTCCGCC
ACACCC SET PGTSESATPESGGG EVQLVESGEG
LVQ
GAAAGTGETGGCGGAGAGGTTCAGCTGGIG GAGTCTGG
CGGIGGCCTGGTGCAGCCAGGGGECTCACTCCGTTIGTCCTETGCA
PGGSLRLSCAASGFNFSSSS I HWVRQAPG K
GCTICTGGCTICAACITITCTICTICTTCTATACACIGGETGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTECAT
CTAMCT GLEWVASISSSYGYMADSVKGRETISADT
TCTrCTTATGGCTATACTTATTATE
CCGATAGCGTCAAGGGCCETTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTA
SKNTAYLQMNSLRAEDTAVYYCAREGSGV
CAAATGAACAGCTTAAGAGCTEAEGACACTGCCGTCTATTATTGTECTCECGGTEETTCTGETETTICTCATTACGETT
CTGITTAC SHYGSVYYSWWALDYWOQGTLVIVSSGG
TACICTTEGIGGECTTTEGACTACTGGGGICAAGGAACCCTGGICACCGTCTCCTCGG GTGGAG
GTGGCAGIGATATCCAGATGA GGS DI QMTQS
PssuAsvG DRVTITCRASQ
CCCAGTCCCCGAGCTCCCTGICCGCCTCIGTGGGCGATAGGGICACCATCACCTGCCGTGCCAGTCAGTCCGTGICCAG
CGCTGTA SVSSAVAWYQQKPGKAPKWYSASSLYSG
GCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTT
CTCG CTTC VPSRFSGSRSGTDFTLIISSLOPEDFATYYC
TCTGETAGCCGTTCCGGGACGEATTICACTCTEACCATCAGCAETCTGCAGCCEEAAEACTTCGCAACTTATTACTETC
AECAAGC QOASYAPITFGCLETKVEIK*

TTCTTACGCTCCEATCACGTTCGGACAGGETACCAAGGIGGAGATCAAA
No en C
0) I-a Ln N) N) I-a No ID DNA
SEQ Protein SEQ
ID

ID to cr, CAGCTTCTGGCTTCAACA 43 EVOIVESG GGLVQPGGSLRLSCAASGEN1G 44 , hole- TCGOTCTTCTTCTATCCACTGGGTG CGTCAGGCCCCEGGTAAGGGCCIGGAATGGGTTG
CATCTATTTATTCTGCTTTTGCCTCTA SSSIHWVRQAPGKG

CTICTTATGCCGATAGCGICAAGGGCCGmCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACA
GCTTA ADSVKG RFTISADTSKNTAYLQMNSLRAED

AGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTACCATTTCCCGTTCGOTTTGCMGGACTACTGGGGICAAGGAA
CCCT TAVYYCARYHFPFGFALDYWGQGTLVTVS
GGICACCGTCTCCICGG GIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTUCCECCICTGTGGG
CGATAGG SGGGG SDI QMTQSPSSLSASVG

GTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCOLGCGCTGTAGCCTGETATCAACAGAAACCAGGAAAAGCTCCGA
AGCTICT ASQSVSSAVAWYQQKFIGKAPKILIYSASSL
GATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTICTCGCTICTCTGOTAGCCGTTCCGGGACGGAMCACFCIGAC
CATCAG YSGVPSR
FSGSRSGTOFTLTiSSLQPEDFAT
CAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGGTETTTACCTGTTCACETTCG
GACAGGGTACCAAGGIGGAG
YYCQQGVYLFTFGQGTKVEIKLEDKTHTKV
ATCAAACTOGAGea case actcacacaAAAGTTGAGCCCAAATCTICTgataagacccataatIGCCCACCGTGCCCAGCACCTGAACTCCT
EPKSSDKTHNCPPCPAPELLG G F'SVFLFP PK
GGGGGGACCGTCAGTMCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGIGG PKDTLMISRTPEVICVVVDVSH EDPEVKFN
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGIGGACGGCGTGGAGGIGCATAATGCCAAGACAAA
GCCGC WYWDGVEVHNAKTKPREEQYNSTYRVVS
GGGAGGAGCAGTACAACAGCACGTACCGTGIGGICAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
VLTVLHQDWLNGKEYKCKVSNKALPAPI E
AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAACCACAGGT
KTISKAKGQPREPQVYTLPPIR ELMTSN QV
GTACACCCTG
CCCCC.AATCCGGGAGCTGATGACCAGCAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGC17CTATCCCAGCGAC
SLSCAVKGFYPSDIAV EW ESN G QPENNY K
ATCGCCGTGGAGTGGGAGAGCAATG G GCAGCCGGAGAACAACTACAAGACCACG

TTPPVLDSDGSFFLVSKLTVDKSRWQQG N

CAGCAGGGGAACGTCTICTCATGCTCCGTGATGCATGAGGCTCTGCA
VFSCSVM HEALHNHYTQKSISISPGKSGSE
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAMAGCGGCAGCGAGACTCCCEGGACCTCAGAGTCCGCCA
CACCC TPGTSESATPESEGGEVQLVESGGGIVQP
GAAAGTGGIGGCGGAGAGGITCAGCTGGTG GAGTCTG G
CGGIGGCCTGGTGCAGCCAGGGGGCTCACTCCGMGTCCTGTGCA
GGSLRLSCAASGFNLSYYYMH WVRQA PG
GCTICTGECTICAACCTCTCTTATTATTATATGCACTGGETGCGICAGGCCCCGGGTAAGGGCCTGGAATEGGITGCAT
CTATTTAT KGLEWVASIYSSYGYTYYADSVKG aril SAD
TCTTCTTATGGCTATACTTATTATG CCGATAG
CGTCAAGGGCCETTICACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTA
TSKNTAYLQMNSLRAEDTAWYCARWSH

TATGGAC VSGHYSGMDYWGQGTLVTVSSGGGGSDI
TACIGGGGICAAGGAACCCTGETCACCGTCTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCT
CCCTGT QMTQSPSSLSASVGDRVTITCRASQSVSSA

GAAACCA VAWYQQKPGKAPKWYSASSLYSGVPSRF
GGAAAAGCTCCGAAGCTTCTGAMACTCGG CATCCAGCCTCTACTCTGGAGTCC
CTTCTCGCTICTCTGGTAGCCGTTCCGGGACG
SGSRSGTDFILTISSLQPEDFATfYCQQSSY
GATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTICGCAACITATTACTGICAGCAATCTTCTTATTCTCTGA
TCACGTTCG SLITFGQGTKVEIV

GACAGGGTACcAAGGTGGAGATCAAA
No C
0) I-a Ln N) N) TABLE LA

ID DNA
SEC! Protein SEQ, ID

ID rnt knob- TCTATTATTCTTCTATCCA CIGGGIGCGTCAGG Cat G GGTAAGGECCIGGAATGGGITG
CATCTATTTATCCTTATTAYGGCTATA
YSSIHWVROAPGKGLEWVASIYPYYGYTYY

CTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAA
CAGCTTA ADSVKGRFTISADTSKIITAYLQMNSLRAED

AGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTACTACCATTACGGITTGGACTACTGGGGTCAAGGAACCCTGG
TCACCGT TAVYYCARYYHYGLDYWGQGTLVTVSSGG
CTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGIC
ACCATC GGSDIQMTQSPSSLSASVGDRVTITCRASQ
ACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGA
TTTACTC SVSSAVAWYMPGKAPKWYSASSLYSG

AGTCTGC.A VPSRFSGSRSGTDFTLTISSLOPEDFATYYC
GCCGGAAGACTTCGCAACTTATTACTGICAGCAATCTTACTGGCATTCTTACCTGATCACGTTCGGACAGGGTACCAAG
GTGGAG A QQSYWHSYLITFGQGTKVEIKLEDKTHTKV
TCAAACTCGAGgacaa a a ctcaca c a AAAGTG GAGCCCAAAACTTCTgata age cc catactIG
CCCACCGTGCCCAECACCTGAACTUTG E
PKTSDKTHTCPPCPAPELLGGPSVFLFP PK
GGGGGACCGTCAGTCTTCCICTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCG
TGGTGGT PKDTLMISRTPEVTCVVVDVSHEDPEVKFN
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCG WYVDGVEVHNAKTKPREEQYNSTYRVVS
CGAGGAGCAGTACMCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT
AC.AA VLTVLHQDWLNGKEYKCKVSNKALPAPIE
oe a GTGCAAGGTCTCCAACAMGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAA
TGGIG KTISKAKGQPREPMVFDLPPSREEMTKNQ
1TrGACCTGCCCCCATCCCOGGAGGAGATGACCAAGAACCAGGICAGCCTGIGGTGCATGGTCAAGGGCTrCTATCCCA
GCGACA VSLWCMVKGFYPSDIAVEWESNGQPENN
TCGCCGTGGAGIGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTT YKTIPPVLDSOGSFFLYSKLTVDKSRWQQG
CCTGTACAGCAAGCTCACCGTGGACAAGAGCCECTGGCAGCAGGEGAACGTCTICTCATGCTCCGTGATGCATGAGGCT
CTGCAC NVFSCSVMHEALHNHYTQKSULSPGKSG
AACCACTACACGCAGAAGAGCCTCTCCCIGTCTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCA
CACCCG SETPGTSESATPESGGGEVQLVESGGGLVQ
AAAGTGGIGGCGGAGAGGTTCAGCTGGIGGAGTCTEGCGGIGGCCIGGTGCAGCCAGGGGGCTCACTCCGTTIGTCCTG
TGCAG PGGSLRLSCAASGFNISYSSIHWVRQAPGK
CTICTGGCTTCAACATCTCTTATTCTTCTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATA
TATTTCTT GLEWVAYISSYYGYTYVADSVKG
RFTISADT
CTTATTATGGCTATACTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGC
CTACCTAC SKNTAYLQMNSLRAEDTAVYYCARAHYFP
AAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTICCCGTGGGCTGGTGCTAT

TGGGGICAAGGAACCCTGGICACCGTCTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGcTcca GTccG MTQSPSSLSASVGDRVTITCRASQ,SVSSAV
CCTCYGTGGGCGATAGGGICACCATCACCTGCCGTGCCAGICAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAA
ACCAGG AWYQQKPGKAPKILIYSASSLYSGVPSRFS
AAAAGCTCCGAACCTICTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTG
GTAGCCGTTCCGGGACGG A
GSRSGTDFILTISSLOPEDFATTYCQQYYW
TTTCACTCTGACCATCAGC.AGTCTGCAGCCGOAAGACTTCGCAACTTATTACTGICAGCAATACTACTGGCCGATCAC
GTTCGGACA PITFGOGTKVEIK=

GGGTACCAAGETGGAGATCAAA

C
0) I-a Ln N) N) I-a TABLE IA

No ID DNA
SEQ Protein SEQ
ID

ID 5 it GAGETICAGCTGGTEGAGICTGGCGGIGGCCTEGTGCAGCCAGGGEGCICACTCCGIUGTCCTGTGCAGCTTCTGGCTI

c hole.
TCTATTATTCTICTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGEGTTGCATCTATTTATCCITATTA
TGGCrATA Y551HWVROPGRGLEWVASIYPYYGYTYY

CTTATTATGCCEATAGCGTCAAGGGCCGMCACTATAAGCGCAGACACATCCAAAAACACAECCTACCTACAAATGAACA
GCTTA ADSVKGRFISADTSKNTAYLQMNSLRAED

CCGT TAVYYCARYYHYGLDYWGCLGTINIVSSGG
CTCCTCGGGTGGAGGTGGCAGTGATATCCAGATGACCCAGICCCCGAECICCCIGTCCGCCTCTGTGGGCGATAGGGIC
ACCATC GGSDICHTOSPSSLSASVGDRVTITCRASQ
ACCTECCGTGCCAGTCAGTCCGTGTCCAGCGCTETAGCCTGGIATOACAGAAACCAGGAAAAGCTCOGAAGCTTOTGAT
TTACTC SVSSAVAWYQQKPEKAPKWYSASSLYSG
GGCATCCAGCCTCTACTCTGGAGTCCCTICTCGCTICTCTGGTAGCCETTCCGEGACEGATUCACTCTGACCATCAGCA
GICTGCA VPSRFSGSRSETOFTLTISSLCIPEDFATVYC
GCCGGAAGAMCGCAACITATTACTGICAGCAATCITACTGGCATTMACCTGATCACGTTCGGACAGGGTACCAAGGTGG
AGA QQSYWHSYLITFGQGTKVEIKLEDKTHTKV
TCAAACTCGAGgacaaaactcacacaAAAETTGAGCCCAMTCTTCTgataagacccataatIGCCCACCEIGCCCAGCA
CCTGAACTCCIG EPIGSDKTHNCPPCPAPELLGGPSVFLFPPK
GGEGGACCGTCAETCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGEACCCCTGAGEICACATECG
TGETEGT PKDILMISRTPEVTONVDVSHEOPEVKFN
GEACGIGAGCCACGAAGACCCTGAGGICAAGTTCAACIGGTACGTEGACGGCGIGGAGGTECATAATECCAAGACAAAG
CCGCG WYVOGVEVHNAKTKPREEQYNSTYRVVS
GGAGGAECAGTACAACAECACGTACCGTGIGGICAGCGTCCTCACCETCCTGCACCAEGACTGECTGAATEGCAAGGAG
TACAA VLTVLHQDININGKEYEKVSNKALPAPIE
GTECAAGGTCICCAACAMGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGEGCAGCCCCGAGAACCAC
AGGIG KTISKAKGQPREPQVYTLPPIRELNITSNQV
TACACCCTGCCCCCAATCCGGGAGCTGATGACCAGCAACCAGGICAGCCTGAGCTGCECCETCAAAEGCTICTATCCCA
GCGACAT SLSCAVKGFYPSDIAVEwEsNGQPENNYK
CECCGTGGAGTEGGAGAGC.AATGGECAECCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGEACTCCGACGGCTC
CTTCTTC TTPPVLDSDGSFFLVSK1.IVOK5RWQQGN
CTCGTGAGCAAGCTCACCGTGGACAAGAGCAGETGGCAECAGGGGAACGTCTICTCATGCTCCGTGATGCATGAGGCTC
TGCACA VFSCSVMHEALHNHYTQKSLASPEKSGSE
ACCACTACACGCAGAAGAGCCTCTCCCTGICTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCAC
.ACCCGA TPGTSESATPESGGGEVQLVESGGGLVQP
AAGTGETEGCGEAGAGGTICAGCTGGIGGAGICTGGCGGTGGCCTEGTGCAGCCAGGGGECICACTCCEITTGICCTET
ECAGC GGSLRLSCAASGFNISSYYIHWVROAPGKG
TTCIGGCTTCAACATCTCTTCTTATTATATCCACTGGGIGCGTCAGGCCCCGGGIAAGGGCCTGGAATGEGTTECATCT
AMATIC LEWVASIYSSYGYTSYADSVKGRFTISADTS
TICTIAIGGCTATACTTCTTATECCGATAGCGTCAAGGGCCETITCACTATAAGCGCAGACACATCCAAAAACACAGCC
TACCTACA KNTAYLQMNSLRAEDTAWYCARTVRESK
AATGAACAECITAAGAGCTGAGGACACTGCCGICTATTATTGIGCTCGCACIGITCGTGEATCCAAAAAACCGTACTTC
TCTGGIT KPYFSGWAMDYWEQETLVIVSSGGEGS
GGGCTATGGACTACTGGGGTCAAGGAACCCTEGTCACCGTCTCCTCGEGTGGAGGTEECAGIGATATCCAGATGACCCA
GTCCCC DIQMTQSPSSLSASVGDRVTITCRASOSVS
EAGCTCCCIGTCCGCCICTGIGGECGATAGGGTCACCATCACCTGCCGTGCCAGTCAGICCGTGTCCAECECTGTAGCC
TGGTATC SAVAWYQQKPGKAPKWYSASSLYSGVPS
MCAGAAACCAGGAAAAGCTCCGAAGCTICTGATTTACTCEGCATCCAGCCTCTACICTGGAGTCCCTICICECTTCTCT
EGTAGCC RFSGSRSGTDFTLTISSLQPEDFATYYCQQY
ETTCCEGGACGGATTTCACTCTEACCATCAGCAGTCTECAGCCGGAAGACTTCGCAACTTATrACTGICAGCAATACTC
TIGGGGT SWGPFTFGQGTKVElIct CCGTTCACGTTCGGACAGGGTAcCAA6GTGGAGATCAAA

C
0) I-a Ln N) N) TAG LE lA

No =
ID DNA
SEQ Protein SEQ
I D

ID

CTGTGCAG CTICTGGCTICAA CA 80 EVQLVESGGGLVO.PGGSLR LSCAASGFN IS 81 knob- TCTCTTCTTCTICTATGCACIGGGTGCGTCAG GC CCC G
GGTAAGGGCCMGAATGGGITGCATCTAMATICTTATTATGGCTCTA
SSSM HAN ROAPGKGLEVVVASIYSYYGSTY

CGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTA
YADSVKGR FTISADTSKNTAYLQMNSLRAE

AGAGCTGAGGACACTGCCGTCIATTATTGTGCTCGCTGGTACGGTATGGACTACTGGGGTCAAGGAACCCTGGTCACCG

GGGTGGAGGTGGCAGTGATATCCAGATGACCCAGTC CCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAG
GGTCACCATCACCTGC GGGSDIQMTQSPSSLSASVG
DRVTITCRAS
CGTGCCAGICAGTCCGTGTCCAGCGCTGTAG
CCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATC
QSVSSAVAWYQQKPGKAPK WYSASSLYS
CAGCCICTACTCTGGAGTCCCTTCTCGCTICTCTGGTAGCCGTTCCGEGACGGATTTCACTCTGACC.ATCAGC.AGTC
TGCAGCCGGA GVPSRFSGSRSGTOFTLTISSLQPEDFATYY
AG ACTTCGCAACTTATTACTGTCAG
CAACCGGGTTCTTGGTACTTCCCGCCGATCACGTTCGGACAGGGTACCAAGGTG GAGATCA
CQQPGSWYFPPITFGQGTKVEKLEDKTHT
AACTCGAGgecaaaactca cacaAAAGTGGAGCCCAAMCTTC1'gatai gacccatactTGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
KVEPKTSDKTHTCPPCPAPELLGGPSVFLFP
GGACCGTCAGICTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCGTGG
IGGTGGA PKPKOTLM ISRTPEVTCVVVDVSHEDP
EVK
CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG
CGCGA FNWYVDGVEVH NAKTKPR EEQYNSTYRV
GGAGCAGTACAACAGCACGTACCGTGTG GTCAG CGTCCTCACCGTCCTGCACCAGGACTGG CTGAATGG
CAAGGAGTACAAGTGC VSVCR/ LH
QDWINGKEYKCKVSNKALPAP
1:1\ AAGGTCTCCAACAAAG
CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAATGGIGTTIG
FE KTISKAKGQPREPMVFDLPPSREEMTK
ACCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGICAGCCTGTCGTG
CATGGTCAAGGGCTICTATCCCAGCGACATCGC
QVSLWCMVKGFYPSDIAVEWESNGQPEN
CGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTICCTG
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
TACAGCAAGCTCACCGTGGACAAGAGCCGCTGGCAGCAGGG GAACGTCTTCTCATGCTCCGTGATG CATE AGG
CTCTG CACAACC GNVFSCSV MHEALHN
HYTQKSLSLSPGKS
ACTACACGCAGAAGAGCCTCTCCCTGICTCCGEGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACC
CGAAAG GSETPGTSESATPESGGGEVQLVESGGGLV
TGGTG GeGGAGAGGTI1CAGCT6ETGGAGIC1GGCGGIGGCCIG

QPGGSLRLSCAASG FNISYSSIHWVRQAPG
GGCTTCAACATCTCTTATTCTTCTATCCACTGGGIGCGTCAGG

KGLEWVAYISSYYGYTYYADSVKGR FTI SAD
TATGGCTATACTTATTATGCCGATAGCGTCAAG
GGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAAT
TSKNTAYLQMNSLRAEDTAVYYCARAHYF
GAACAGCTTAAGAGCTGAGGACACTGCCGICTATTATTGTGCTCGCG CTCATTACTTCCCGTGGGCTGGTG
CTATGGACTACTGGG
PWAGAMDYWCQGTLVTVSSGEGGSDI Q
GTCAAGGAACCCIGGICACCGTCTCCTCGGGIGGAGGIGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTC
CGCCTCT M TO$PSSLSASVGDRVTITC
RASQSVSSAV
GTGGGCGATAG GGTCACCATCACCTG CCGTGCCAGTCAGTCCGTGTCCAG
CGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAG
AWYQQKPGKAPKWYSASSLYSGVPSRFS
CTCCGAAGCCTGATTTACTCGG CAT CCAGCCTCTA CTCTG G AG TCCCTTCTCGCTTCT CTGGTAG
CCGTTCCGGGACGG ATTICA GSRSGTD
FTLTISSLQPEDFATYYCQQYYW
CTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACTACTGGCCGATCACGTTCGG

ACCAAGGTGGAGATCAAA
No en C
0) I-a Ln N) N) I-a No ID DNA
SEQ Protein SEQ
ID

ID ot hole-TCTCTTMCTTCTATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCIATTIATTCTIATTATG
GCTCTA SSSNIHWVROAPGKGLEWVASIYSYYGSTY

CITATTATGCCGATAGCGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAPACACAGCCIACCIACAAATGAA
CAGUTA YADSVKGRFTISADTSKNTAYLQMNSLRAE

AGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCIGGTACGGTATGGACTACTGGGGICAAGGAACCCTGGICACCG
ICTCCTC DTAVYYCARWYG MDYWG OGILVTVSSG
GGGIGGAGGTGGCAGTGATAICCAGATGACCCAGTCCCCGAG
CTCcaorccGCCTCTGTGGGCGATAGGGIC.ACCATCACCTG C GGSSDIQMIQSPSSI.SASVG

CGIGCCAGTCAGTCCGTGTCCAGCGCTGIAGCCTGGTATCAAC.AGAAACCAGGAAAAGCTCCGAAGCTICTGAMACIC
GGCATC CISVSSAVAWYQQKPG KAP KWYSASSLYS
CAGCCICTACTCTGGAGTCCCITCICGCITCTCTGGTAGCCGTTCCGGGACGGATTICACTCTGACCATCAGCAGTCTG
CAGCCGGA GVPSRFSG SRSGTOFTLTISSLQPEDFATYY
AGACTICGCAACTTATTACTGTCAG
CAACCGGGTICTIGGTACTICCCECCGATCACGITCGGACAGGGIACCAAGGTGGAGATCA
CQQPGSWYFPPITFGQGTKVE I KLEDKIHT
AACCGAGgacariaactca cacaAAAGTTGAGCCCAAATCTTCTgeta egacccataetTGCCCACCGTGCCCAGCACCTGAACTCCIGGG G KVERSSDKTHNCPPCPAPELLGGPSVFLF
GGACCGTCAGTCTTCUCTTCCCCCCAAAACCCA/kGGACACCCTCATGATCTCCCGGACCCCTGAGGICACAIGCGTGG
TGGIGGA PPKPKDTLMISRTPEVICVVVDVSHEDPEV
CGTGAGCCACGAAGACCCTGAGGICAAGTTCAACTGGIACGTGGACGGCGIGGAGGTGCATAATG CCAAGACAAAG
CCGCGG GA KFNWYV DGVEVH NAKTKP R EE QYN STf R
GGAGCAGTACAACAGCACGTACCGTGIGGICAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATEGCAAGGAGTACAAGTGC VVSVITVLFIQDW LNG KEYKCKVSNKALPA
AAGGICTCCAACAAAG CCCICCCAGCCCCCATCGAGAAAACCATCTCCAAAG CCAAAGG G
CAGCCCCGAGAACCACAGGTGTACA PI EKTISKAKGQPREPQVYTLP PI RELMTSN
CCCTGCCCCC.AATCCGGGAGCTGATGACCAGCAACCAG

ESNG QPE NN
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCITMCCTCG YKTIPPVLDSOGSFFLVSKLTVDKSRWQQ
TGAG
CAAGCTCACCGTGGACAAGAGCAGGIGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CA GNVFSCSVMHEALHNHYTQKSLSLSPGKS
CTACACGCAGAAGAGCCTCTCCCIGTCTCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCC
GAAAGT GSETPGTSESATPESGGGEVCILVESGGGLV
GGIGGCGGAGAGGTICAGCTGGIGGAGTCTG
GCGGTGGCCTGGIGCAGCCAGGGGOCTCACTCCGTTTGICCTGTGCAGCTICT
QPGGSLRLSCAASGFNISSYYIHWVROAPG
GGCTICAACATCTCTTCTTATTAIATCCACTGGGIGCGTCAGG

TATGG CTATACTICTTATGCCGATAGCGTCAAGGGCCGTTICACTATAAG
CGCAGAC.ACATCCAAAAACACAGCCTACCTACAAAT TSKNTAYLQMNSLRAEDTAVYYCARTVRG
GAACAGCTTAAGAGCTGAGGACACTGCCGICTATIATTGTGCTCGCACTM
CGTGGATCCAAAAAACCGTACTICTCTGGTTGGG SKKPYFSGWAMDYWGQGTLVIVSSGGG
CTAIGGACTACTGGG GTCAAGGAACCCIGGTCACCGICICCTCGGGIG
GAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAG GSDIQMIQSPSSLSASVGDRVTITCRASQS
CTCCCTGICCGCCTCTGTGGGCGATAGGGTCACCATCACCIGCCGTGCCAGTCAGTCCGTGICCAGCGCTGTAGCCTGG
IATCAAC VSSAVAWYQQKPG KAPKWYSASSLYSGV
AGAAACCAGGAAAAGCTCCGAAGCTICTGATTTACTCGGCATCCAGCCTCTACICTGGAGTCCCITCTCGCTICTCTGG
IAGCCGIT PSRFSGSRSGTDFILTISSLCIPEDFATYYCQ
CCGGG
ACGGATTTCACTCTGACCATCAGCAGICTECAGCCGGAAGACTTCGCAACTTATIACIGTCAGCAATACTCTTGGGGIC
CG QYSW6PFTFGQ6IKVE I K* 1-3 TTCACGTTCGGACAGGGTACCAAGGIGGAGATCAAA
No tio C
0) I-a Ln N) N) TAB LE 1.A

No ID DNA
SEQ Protein StQ
ID

ID ot knob- CCTUTTTTATITTATG
CACTGGETGCGTCAGGCCCCGGGTAAGGGCCIGGAATGGGTTGCAACTGITTATCCTTATCTTGACTATA
FYFM HWVROAPG KGLEWVATVYPYLDYT

CGTCAAGGGCCGITTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAMTGAACAGCTTA
YYADSV KGRFT ISA DISK NTAYLQM NS LRA

ACTACTGGGGT CAAGG AACCCT
EDTAVYYCARAFPGSYHPMDYWGQGTLV
GETCACCGTCTCCTCGGGIGGAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGUCCCIGTCCGCCICTGTGG
GCGATAG G
TVSSGGGGSDIQMTQSPSSLSASVGDRVTI
GTCACCATCACCIGCCGTGCCAGICAGICCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGA
AGCTTCT TCRASQSVSSAVAWYQ0J(PG KAP
KLUYSA
GATTTACTCGGCATCCAGCCICTACTCTGGAGTCCCTICTCGCTTCTCTGGTAGCCGTTCCGGGACGGAMCACTCTGAC
CATCAG
SSLYSGVPSRFSGSRSGTOFTLTISSLOPEDF
CAGICTGCAGCCGGAAGACTICGCAACTMYTACTGTCAGCAATCTIVITATTCTCTGATCACGTTCGGACAGGGTACCA
AGGTGG ATYYCQQSSYSLITFG QGTKVEI
KLEDKTHT
AGATCAAACTCGAGgaceanactca cacaAAAGIGGAGCCCAMACTICTgataagacccatactIGCCCACCGTGCCCAGCACCrGAACTC
K V EPKTSDKTHTCP PCPAPELLGGPSVFLF P
CTGGGGGGACCGICAGTCTICCTCTICCCCCCAAAACCCAAGGACACCCICATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGT PKPKDILMISRIP EVTCVVVDVSH
EDPEVK
GGTEGACGIGAGCCACGAAGACCCTGAGGICAAGTICAACTG
GTACGTGGACGGCGTGGAGGIGCATAATGCCAAGACAAAGCC
FNWYVDGVEVHNAKTKPRE EQYNSTYRV
GCGCGAGGAGCAGTACAACAGCACGTACCGTGTGGICAGCGICCTCACCGTCCTGCACCAGGACTGaTGAATGG
CAAGGAGTA
VSVLTVLHQDWLNGKEYKCKVSNKALPAP
a oo CAAGTGCAAGG/CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAMACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC

GIGTTTGACCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGICAGCCTGIGGTGCATGGTCAAGGGCTTCTATC
CCAGCG QVSLWCMVKG FYPS DIAV EWES NGQP
EN
ACATCGCCGTEGAGIGGGAGAGCAATGGG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGIGCTGGACTCCGACGGCTCCT
NYKTIPPVLDSDGSFFLYSKLIVDKSRWQQ
TCTICCIGTACAGCAAG
CTCACCGTGGACAAGAGCCGCTGGCAGCAGGGGAACGICTICTCATGCTCCGTGATGCATGAGGCICT
GNVFSCSVM HEA L FIN RYTQKSLSLSPGS
GCACAACCACTACACG CAGAAGAGCCTCTCCCIGTCTCCGGGTAAAAGCGG CAGC GAGA CTCCCGGGACCTCAG
AGTCCG CCAC A G SETP G TSESATP
ESGGGEVQLVESGGG LV
CCCGAAAGIGGTGGCGGAGAGGTTCAGCTGGTGGAGTCTGGCGGIGGCCTGGIGCAGCCAGGGGGCTCACTCCGTITGT
CCTGT QPGGSLRLSCAASG FNISYSSI HWV R
QAPG
G CAGCTICIGGCTTCAACATCICTTATTCTTCTATCCACTGGGTGCGTCAGGCCCCGGGTAAGG
GCCTGGAATGGGTTG CATATAT
KGLEWVAYISSYYGYTYYADSVKGRFTISAD
TTCTTCTTATTATGGCTATACTTATTATGCCGATAGCGTCAAG
GGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTA
TSKNTAYLQMNSLRAEDTAVYYCARAHYF
CCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTCATTACTICCCGTGGGCTGGT

ACTACTGGGGTCAAGGAACCCIGETCACCGTCTCCTCGGGTOGAGGIGGCAGTGATATCCAGATGACCCAGICCCCGAG
CTCCCT MTQSPSSLSASVGDRVTITCRASQSVSSAV

CAGAAAC AWYQQKPGKAPKLLIYSASSLYSGVPSRFS

CCGGGA GS MGT DR' LTISSLQP
EDFATYYCQQYYW
CGG ATTr CACTCTG ACCATCAGCAGTCTGCAGCCGGAAGA CTTCGCAACITATTACTGICAG CAATACTACTG
GCCGATCACGTTCG PITFGQGTKVEIK*

GACAGGGTACCAAGGTGGAGATCAAATGA
co No ea.
tit C
0) I-a Ln N) N) No ID DNA
SEQ Protein SEQ
I D

I D 5 it hole- CCTUTMATTTTATG CACT GG GTGCG TCAG GC CCCGG G TAAGG G C CTG GAATGGGTTG
CAA CTGITTATC CTTATCTTGACTATA FYFMHWV ROAPGKG LEW VATVY PY
L DYT

CCTACCTACAAATGAACAGOTA YYADSVKG
RFTISADTSKNTAYLQMNSLRA

CTCGCGCGTITCCGGGITCTTACCATCCTATEGACTACTGGGGICAAGGAACCCT
EDTAWYCARAFPGSYHPM DYWGQGTLV
GGICACCGTCTCCTCGGGTGCAGGTGGCAGTGATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGIGGGC
GATAGG TVSSGGGG SDI
QMTOSPSSLSASVGDRVTI
GTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGA
AGCTTCT TCRASQSVSSAVAWYQQKPGKAPKWYSA
GATUACTCGGCATCCAGCCTCTACTCTGGAGTCCCTICTCGCTTCTCTGGTAGCCETTCCGGGACGGATTTCACTCTGA
CCATCAG SSLYSGVPSRFSGSRSGTDFTLTI SSLQP
EDF
CAGTCTGCAG CCGGAAGACTTCG

ATYYCQQSSYSLITFGQGTKV El KLEDKTFIT
AGATCAAACTCGAGga ca a aactcaca caAAA GTTG AG CCCAAATCTTCTpta aga =ate atTG CC

PSVFLF
CTGG G
GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGICACATGCGTG
GT PPKPKDTLM ISRTPEVTCVVVDVSHEDPEV
GGTGGACGTGAGCCACGAAGACCCTGAGGICAAGTTCAACTEGTACGIGGACGECGTGGAGGTG CATAATG
CCAAGACAAAG CC
KFNWYVDGVEVHNAKTKPREEQYNSIYR
GCGGGAGGAG CAGTACAACAGCACGTACCGIGTG GTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTA
VVSVCRILHODWLNGKEYKCKVSNKALPA
CAAGTGCAAGETCTCCAACAAAGCCCTCCCAGCCCCCATCGAG'AAAACCATCTCCAAAGCCAAAGGECAGCCCCGAGA
ACCACAG PI EKTISKAKGQPREPQVYTLPP IRE
LMTS N
GIGTACACCCTGCCCCCAATCCGGGAGMATGACCAGCAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCC
AGCG QVSLSCAVKGFYPSDIAVEWESN GQPENN
ACATCGCCGTGGAGTEGGAGAGCAATGGG
CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACrCCGACGGCTCCT
YKTTPPVLDSDGSFFLVSKLTV DKSR WOO
TCTTCCTCGTGAG CAAGCTCACCGTGGACAAGAGCAGGIGGCAGCAGGGGAACGICTTCTCATG

NHYTQKSLSLSPG KS
GCACAACCACTACACG
CAGAAGAGCCTCTCCCIGTUCCGGGTAAAAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACA
GSETPGTSESATPESGGGEVQLVESGGGLV
CCCGAAAGTGGTGGCGGAGAGGITC.AGCTGETGGAGTCTGGCG
GTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCIGT
QPGGSLRLSCAASGFNISSYYI HWVROAPG
GCAG CTTCTG G CTTCAACAT CT CTTCTTATTATATCCACT G G GTG CG TCAGG CCCCGG GTAAG G
G C CTGGAATGGGTTG CAT CTAT
KGLEWVASIYSSYGYTSYADSVKGRFTISAD
TTATTCTICTTATGGCTATACTTUTATGCCGATAG CGICAAGGGCCGTITCACTATAAGCG
CAGACACATCCAAAAACACAGCCTA
TSKNTAYLOPINSLRAEDTAVYYCARTVRG
CCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGIGCTCGCACTGTTCGTGGATCCAAAAAACCG
TACTTCTC SKKPYFSGWAMDYWGQGTLVTVSSGGG
TOG TTEGGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTC CTCGGGTGGAGGTG
GCAGTGATATCCAGATGACCCAG GSD I
QMTQSPSSLSASVGDRVTITCRASQS
TCCCCGAGCTCCCTGICCGCCTCTGIGGGCGATAGGGTC.AcCATCACCTG
CCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTG
VSSAVAWYQQKPG KAPK LLIYSASSLYSGV
GTATCAACAGAAACCAG GAAAAG CTC CGAAG CITCTGATTTA CTCG G CATCCAGCCICTA CT CTG
GAGTCCCTTCTCGCTTCTCTGG
PSRFSGSRSGTDFTLTISSLO,PEOFATYYCQ
TAGCCGTICCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACITATTACTGTCAGCAA
TACTCTT QYSWGPFTFGQGTKVEIK*

GGGGTCCGTICACGTTCGGACAGGGTACCAAGGTGGAGATCAAATGA
No C
U) A
A

Ln N) N) 17' 1--, Table 12 r.) w CD
be Cs b.) o ID N- ISM HQ VII-VL N- SEQ SEQ Ft fusion C- 5ECI SEQ VH-V1. C- SEQ
riti ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NO: VL NO: NO:
VII NO; NO: VL NO:
(V111) (VII) (VH3) NIA

CTCGAGiaceaastNacataAAAGTGGAGCC 2539 49 GAGGTTCAGCTGGTG 46 GGTGGA 2542 knob- TGGAGICTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACITCTosagaccsatactTGCCC.ACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTOCCCAGCACCTGAACTCCIGGGGGGAC CTGGIGCAGCCAGGG ACT
AGCTCCCTGTCC

CGTCAGTCTTCCTOICCCCCCAMACCCA GGCTCACTCCGMGT
GCCICTOGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTE CCTGIGCAGCTICTGG
GATAGGGICACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGICACATGCGTGGIGGTGGACGTGAGC CTICAACATCTaTATT
ATCACCTGCCGT
TCAACATCGGTFCT GICAGTCCETGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG CTTCTATCCACTGGGT
GCCAGTCAGTCC
TCTTCTATCCACTG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAAIG GCGTCAGGCCCCGGG
GIGTCCAGCGCT
GGTGCGTCAGGCC aGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGICC GGITGCATATATTTCT
CAACAGMACCA

TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
c, C ATCTATTTATTCTG TGATTTACTCGGC

AAGCTTCTGATT
CITTTGCCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TCTTATGCCGATA TCTGGAGTCCCIT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGGTGITTGACCIGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTTCT
TTTCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTUGGISCATGGICAAGGGCTICTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CIGACCATCAGCA
CCCAGCGACATCGCCETEGAGTGGGAGA GGACACTGCCGICTAT
ACGGAMCACT
CTACAAATGAACA GICTGCAGCCGG
GC.AATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTIAAGAGCTGA MGACTTCGCAAC
GACCACGCCTCCCGTGCTGOACTCCGACG ATTACTTCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTTCTTCCTGTACAGCAAGCTCACCG TGGTGCTATGGACTAC
GAAGACTTCGCA .
TATTATTGTGCTCG CAAGGTGTTTACC
TGGAC.AAGAGCCGCMGCAGCAGGGGAA TGGGGICAAGGAACC
ACTTATTACTGTC
CTACCATTTCCCGT TETTCACGITCGG
CGICTICTCATGCTCCGTGATGCATGAGGC CTGGICACCGTCTCCT
AGCAATACTACT
TCGGITTTGCTTIG ACAGEGTACCAA
TCTGCACAACCACTACACGCAGAAGAGCC CG
GOCCGATCACGT
GACTACTOGGGTC GGTGGAGATCAA
TCTCCCTGICTCCGGGTAAAAGCGGCAGC
TCGGACAGGGIA
li AAGGAACCCIGGT A
GAGACTCCCEGGACCICAGAGTCCGCCAC
CCAAGGIGGAG n CACOGICTCCTCG
ACCCGAAAGTGGTGGCGGA
ATCAAATGA

be C:, b.) S...:1 c In cm t ca C
U) A
A

Ln N) N) 17' A
r.) Table 13 w be Co cs i--.7 ID hi- SEQ SEQ Vii-VI. JN- SEQ SEQ Fc fusion C- 'SEQ HQ VIM C- SEQ
ia fusion ID ID linker fusion ID ID
fusion ID ID linker fusion ID
WI NO: NO: VL NO: NO:
VII NO: NO: VL NO:
(VIII) (V12) 0413) (VW

CTCGAGgacsasactcacacsAAAGTTGAGCC 2542 53 GAGGTTCACCIGGIG

hole- TGGAGTCTGGCGG GGTGGC ACCCAGTCCOCGA
CAAATCTICTgatsagacccataatTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AGT
AGCTCCCTGTCC
2542 CCAGGGG6CICAC aCTGTGGGCGA
CGTCAGICTTCCTCTTCCCCCCAAAACCCA GGCTCACTCCGTTEGT
GCCTCTGIGGEC
TcCG1T73TCCT3T TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGOTCTGG
GATAGGGICACC
GCAECTICTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGTGGTGGACUGAGC CTTCAACATCTCTICTT
ATCACCTGCCGT

CACGAAGACCCTGAGGICAAGTTCAACTG ATTATATCCACTGGGT
GCCAGTCAGTCC
TCTTCTATCCACTG CAGCGCTGIAGC

GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCIGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC MACCAGGAAAA
CAACAGCACGTACCGTGTEETCAGCGTCC GGITGCATCTATTTAT
CAACAGAisACCA
TGGAATGGGTTGC GCTCCGAAGCTTC

Gfi0AAAGCTCCG
µ2, GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTECTTATGCCGATAG
AAGCTICTGATT
CTITTGCCTCTACT ATCCAGCCICTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TaTATGCCGATA laGessrccCrr =CCATCTCCAAAGCCAAAGGGCMCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CICGCTTaCTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
sesksitccrreT
TTTCACIATAAGCG TAGCCGTICCGO
CCOGGAGCTGATGACCAGCAACCAGGICA CCTACCTACAAATGAA
CGCTICICIGGI
CAGACACATCCAA GACGGAMCACT
GCCTGAGCTGCGCCGTCAAAGGCTICTAT CAGCTTAAGAGCTGA
AGCCGTICCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCCTGGAGTGGGAGA GGACACTGCCGICIAT
ACGGATTTCACT
= CTACAAATGAACA
GICTGCAGCCGG ECAATGGGCAGCCGGAGAACAACTACAA
TATTGTGCTCGCACTG CTGACCATCAGC
GCTTAAGAGCTGA AAGACT/CGCAAC
EACCACGCCICCCGTGaGGACTCCGACG 77CGTGGATCCAMAA
AGICTGC.AGCCG
GGACACTGCCGTC TTATTACTGICAG
GCTCCTICTICCTCGTGAGCAAGCTCACCG ACCGTACTICICTGGI
GAAGACTTCGCA
TATTATTGTGCTCG CAAGGIGITTACC

ACTIATTACTGTC
CTACCATTTCCCGT TGTTCACGTTCGG
CGTCTTCTCATGCTCCGTGATGCATGAGGC GGGGICAAGGAACCC
AGCAATACTCTI
TCGGITTTGaTTG ACAGGGTACCAA
TCTGCACAACCACTACACGCAGAAGAGCC TGGICACCGICTCCTC
GGGGIECCGrfCA
GACTACTOGGGIC GGIGGAGATCAA
TCICCCTGICTCCGGGIAAAAGCGGCAGC G
CGTTCGGACAGG li AAGGAACCCTGGT A
GAUCTCCCGGGACCICAGAGTCCGCCAC
GTACCAAGGIGG n =
CACCGTCECCTCG
ACCCGAAAGTGGTGGCGGA AGATCAAATGA

:

be Co S....:' cs en Los t LH

en in c.

el ei el @
VDIVVVDLVDV

ci) 991.99VVDOVI9 V

Am DDV)V9D0119D
VV)/VDVDDIDD DIDDDDIDV/DV9 VVDDVIDD9VDV Dat391111991.
IL)13V1VV3DV 13D/D/D)DVD/SOL3 $9,119N)1191 9D33.U.DOW13 DiaLDVIIVJJ.OV 33Y09v01$991 VD93113VDVVD 'WYSS/V/391991 9VVVDDMVOV109)DISVDVDIDDVDt9 9401910V1iVII

91)DV)DIDIDV DDDSIDODOILIVIIV
33ai3Y9Y939VDSDD9aggetlis345411 3VVODDIEDVDW
%/01,19VDVVIIDD
D9VDIVDDV910 3109DDDIDDLDI/Vi ommleteellithenpeannceppl2 DDDDDVDDIDID
YDVVD1VVV3V).3 INDLLIV9D3V LY/2ID30:910VDVD9 ettgriravaignmAbvennefiel tODYJIV.IDV9ID
DVIODDV)VDVVV

111499eileleoeferiespheaneppn nvaiivsSay$
YVDDIVOYDvDvD
ISDAD131.0DD WD/VVV3V13OVIDD
Int3zolthidb2peUptibmogonnlio 9930.U.9339V1 ODDVVIVIDVDLU.
IDLLIDOLDVDD i9VDVDVVVVV3311/DV
enpmeellitaban2923eraelentillo 9D41310901,) 1.113V11039V DVDVDDODVVIVIDV
214X113143eine=v1.3Pnee442433 1130319V99/DI

Smetp:943191emedemelielt8Uel1 yvilmsvoniv LIVIlDn 1 III) 11491111,3910V OV1V9DODIVIIVJ.13 moseznamanaltarnotelle93:339 ogonyjnysi DIOLLVILivIDIV .. ei 0.µ
DDDIDDVINVDD YLVID99.LVIIV1J-DI
eAllemliempze33eiedelatexim lupprionop DUI-VD.Ivvv..i1OVV) 13111VIVIVDDL1D9 1s3maniesenenm2gee3912eveaell vVVVODVDDVW

IYIDDLI3DVI9 9/VV$9133$99ra hvileavvt4gilveneX=1411013:01DA DVOVVDIVISO/D
DDS9V)IDDSLDD
LIDDDYDOISID St DD7D799VDI9D9 1311 3111939;133011zolenenzlefiellel DM/1913939V) 9I3VDDIV.01101 1,139331emilenaten139193142149103 319100319V319 IDDODIDDVOLV 1/VIIDIILVDVV31.13 Elliman ea p9e eznit23309. et e3 yx01,93)91my 1.199.01109VDD
30V1199DViv5 D91D.U.D9V3DI.D.00 nel423aih22in2)2log2Ss143333en 31%03VDID9DVi DDDDIDIDIDDO ID.U.IDODINDDDD :mei in mavelle em e ee exams= VS3DDDIDID13 MOIDD9DDSvDD
DILDIDDDIDDV /DV DID9VDDDV)D19910 axa):19041,,elifillpop1013313111273 nousbonioD I9V

DD)33191/303V ODDI9D 39DI9D3991DI9VD
9111133m311113eIme2ev41121D1131VWD VDODDDIDVDD3V 3991D9 DODDOIDIDVDDI ...3 33DVDJJ.9YVV4313uPEgte3a9V9IL3 IS asvevaaivivp it so VDD1DD 90 99/0Dva9tve S4 6105 .6109 (IIA) (IW) (VIA) ON
10N 1A :ON :ON NA :ON
:ON 1/1 :ON :ON HA
01 1.101111) Mull 01 CI voIsill a! GI uvlsni im100 CH al 1.1019111 %a

14 03S .3 1AilA WS ON 1 volgri 33 035 b3S -1,1 'MO WS 133S
=N Cl el el C:
el =

m A
9T allqvI

,--1 N

M
Lc-) 0!

C
U) A
A

Ln N) N) 17' 1--, Table 18 r.) w C
be cic No o ID N- SEQ SEQ VH-VL N= HQ SEQ Fc fusion C- SEQ SEQ VII-VL re Oa tit ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NCH VI NO: NO:
VII NO: FKI: V1 NO:
(11H1.1 (VIA
(1113) (VIA
5019- 5019 45 GAGG1TCAGCT6G 46 GGTGGA 5019 47 Si.
CTCGAGiscassactuicicsAAAGITGAGCC 2542 53 GAGGTTCAGCTGGTG
46 OGTGGA 2542 50 GATATCC.AGATG

CAAATCTICTptaagacccatacttecccsan GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

cccapactipactixtuaggaccitcagtatcct CTGGTGCAGCCAGGG AGT
AGCTCCCTGICC
CCAGGGGGCTCAC GCTCCCTGTCCGC
ctIccccccsaa soccaaggacaccctratgatciccc GGCTCACTCCGTTIGT
GCCTCTGIGGGC

Itta0cecttantatatteliggigginargtbs5c CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT TAGGGTCACCATC
cacpagaccagaggtcaagticaacteetacgtgga CTICAACATCTCTICTT
ATCACCTGCCGT
TCMCATCGGTTCT ACCTOCCGTGCCA
cgmtggaggtgcstastgcceagacsanccgcgg ATTATATCCACTGGGT
GCCAGTCAGICC

pgpscagtacaacagcacitaccgtgtutcagcg GCGTCAGGCCCCGGG
GTOTCCAGCGCT
GGIGCGICAGGCC CAGCGCTGTAGC
tcctoaccecctecaccaggactuctgaatucaag TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCG GGTAAGGGCC CTGGTATCAACAG
gagtacaagtgcsaggtaccascasaguctcccag GOTTGCATCrATITAT
CAACAGAAACCA
TGGAATGGGITGC AAACCAGGAMA
acco1cppaaitcatctccisagccameggps TCTICTTATGGCTATA
GGAAAAGCTCCG
c, ca ATCTATTTATTCTG GCTCCGAAGCTTC
sccccgagoaccscsulgtacaccctscccccatac CTTCTTATGCCGATAG
AAGCTICTGATT
CMTGCCICIACr TGATTTACTCGGC
ogeggeptgaccaspaccaystcartreccte CGICAAGEGCCGITTC
TACTCNCATCC
ICUATECCGATA ATCCAGCCTCTAC
cctsatcaasuctictatcccatcgacatcgccgtgg ACTATMGCGCAGAC
AGCCTCTACTCT
, GCGTCAAGGGCCG TCTGGAGTCCCTT
agtguagagclatgucasccggagaacaactica ACATCCAAAAACACAG
GGAGTCCCTICT
TTTCACTATAAGCG CMGCTTCTCTIG
agaccecgcctcccescluactccgacuctcettet CCTACC7ACAAAT5AA
CGCTICTCTGGI
CAGACACATCCAA TAGCCGTICCGG
tcctictacogcsagcicacalgigacaagaragstag CAGCTTAAGAGCTGA
AGCOGTTCCGGG
AAACACAGCCIAC GACGGATTTCACT
cagtaggsgaacgtottritatsctccistatcacga GGACACTGCCGTCTAT
ACGGATITCACT
CTACAAATGAACA CTGACCATCAGCA
ggctctracaaccactacacgcagaagagutctccc TATTGIGCTCGCACT6 CTGACCATCAGC
GCTTAAGAGCTGA GTCTGCAGCCGG
trctixlIEStaasAGCGGCAGCGAGACTCCC TTCGIGGATCCAAAAA
AGICTGCAGCCG
GGACACTGCCGTC MGACTTCGCAAC
GGGACCTCAGAGTCCGCCACACCCGMAG ACCETACTICTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG TTATTACTGTCAG
TGGTGGCGGA TGGGCTATGGACTACT
ACITATTACTGTC
CTACCATTTCCCGT CAAGGIGTITACC
GGGGTCAAGGAACCC
AGCAATACTACT
TCGGTTITGCMG TGITCACGTTCGG
TGGICACCGTCTCCTC
GGCCGATCACGT
GACTACTGGGGTC ACAGGGTACCAA
G

li AAGGAACCCIGGT GGTGGAGATCAA

CCAAGGTGGAG n CACCGTCTCCTCG A

ATCAAATGA

be C:, No 5-1:1 c LA
cm t Go C
U) A
A

Ln N) N) 17' A
r.) Table 28 w t4 Cs t.) o i--.3 N- SEQ SEQ VII-VL N- SEQ HQ Fausion C- SEQ SEQ VH-VI. Cr SEQ
rat ia fusion ID ID linker fusion ID ID
fusion ID ID linker Won ID
VH NO; NO; Vt. NO; NO:
VH NO; NO; VL NO;
(V111) (VU) On131 (VIA

CTCGAGgscaaaectcsocaMAGTGGAICC 2539 49 GAGGTTCAGCTGGTG 45 GGTGGA 2541 50 GATATCCAGATG
knob- TGGAGICTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACTICTiptaigaccestactIGCCOICC GAGICIGGCGGIGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AGT
AECTCCCTGICC

CGICAGICTICCICTICCCCCCAAAACCCA GGCTCACTCCGITTGT
GCCICTGTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTICTGG
GATAGGGTCACC
GCAGCTMTGGCT ACC'TGCCGTGCCA
AGGICACATGCGIGGTGGTGGAOSTGAGC CITCAACATCTCTTATT
ATCACCTGCCGT
TCAACATCTCTTAT GTCAGICCGTGIC
CACGAAGACCCTGAGGICAAGITCAACTG CTICTATCCACTGGGI
GCCAGICAGICC
TATTATATGCACTG CAGCGCIGTAGC
ETACGTGGACGGCGTGGAGGIGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGIGGICAGCGTCC GGTTGCATATATTICT
CAACAGAAACCA
TIGGAATGGGTTGC GCMCGAAGCTTC
ICACCGTCCTGCACCAOCIACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
.0 4. ATCTATTTATTCTT TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGETCTCCAA CTTATTATGCCGATAG
AAGCTICIGATT
ATTATGGCTATALlu ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTIC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCMAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTICTCTOG
GAACCAATGGTGTrGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTTCT
TTTCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGTCA CCIACCTAC.AAAIGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGIGGTGCATGGICAAGGGCTTCTAI CAGMAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCOGTGGAGTGGGAGA GGAC.ACTGCCGTCTAT
ACGGATTTCACT
CIACAAATGAACA GICTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCGCM
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACrfCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGMAG
GCTCCUCTICCTGTACAGCAAGCTCACCG IGGTGCTATOGACTAC
GMGACTTCGCA
TATTATTGTGCTCG CAACATCCGTGGT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGSTCAAGGAACC
ACTTATTACTGIC
CICTICMCICTT CIGGIGGITACCT
CGTCTICICATGCTCCGTGATGCATGAGGC CIGGICACCGICICCT
AGCAATACTACT
GCGCTATGGACTA GATCACGTTCGG
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGaGATCACET

TCMCCTGTCTCCGGGTAAAAGCGGCAGC
TCGGACAGGGTA
li ACCCTGGTCACCG GGIGGAGATCAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCPAGGIGGAG n TCTCCTCG A
ACCCGAAAGTGGIGGCGGA
ATCAAATGA

t4 C:, S...:' c In cm t coo C
U) A
A

Ln N) N) 17' .--, Tablets r.) w be Co No o L.Z.3 ID N- SEQ SEQ VH-VL N- SEQ SEQ Fc fusion C. SEQ SEQ VII-VL C- SEQ
rits ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NO: VI. NO: NO:
VH NO: NO: VL NO:
IVHII (VU) (VH3I (VIA) .

CTCGAGoacoaaacteaucaAAAGTTGAGCC 2541 53 GAGGTTCAGCTGGTG 46 GGTGGA 2539 52 GATATCCAGATG.
hole- TGGAGTCTGGCOG GGTGGC ACCCAGTCCCCGA
CAAATCTItTptiogicccateatTGCCCACC GAGICTGGCGGIGGC
GGTGGC ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCIGGGGGGAC CTGGTGCAGCCAGGG AGT
AGCTCCCTGIcc . 2542 CCAGGGGGCTCAC
CTCTGTGGGCGA
CGTCAGICTTCCTCTICCCCCCAAAACCCA EGCTCACTCCGTTTGT GCCICTGTGGGC
TCCGTTTETCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTICTGG
SATAGGGICACC
GCAGCTICTGGCT ACCTSCCGTGCCA
AGGTCACATECGTGGIGGIGGACGTGAGC CTTCAACATCTCTTCTT
ATCACCFGCCGT
TCAACATCTCTTAT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG ATTATATCCACTGGGT
GCCAGTCAGTCC
TATTATATGCACTG CAGCGCTGTAGC
GTACGTOGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGEG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGIATCoAcAr.
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT

CAACAGCACGTACCGTGTGGTCAGCGTCC GGITSCATCTATTTAT
CAACAGAAACCA
TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTICTTATOGCTATA
GGAMAGCTCCG
c, ATCTATTTATICIT TGATUACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTCTTATGCCGATAG
AAGOICTGATT
La ATTATGGCTATACT ATCCAGCCTCTAC

TACTCGGCATCC
TATTATGCCGATA ICTGGAGTCCUT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTICT
MCACTATAAGCC TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAAATGAA
CGCTICTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGIGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
ECAATGEGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGaGGACTCCGACG TTCGTGGATCCAAAAA
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTTCUCCTCGTGAGCAAGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG CAACATCCGTGGT
TGGACAAGAGCAGGTGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTTATTACTGTC
CTCTTCMCTCTT CTGGTGGTTACCT
CGTCTTCTCATGCTCCGTGATGCATGAGGC GOGGICAAGGAACCC
AGCAATACICTI
GGGCTATGGACTA GATCACGTTCGG
TCTGCACMCCACTACACGCAGAAGAGCC TGGTCACCGTCTCCTC
GEGGICCOTTCA
CIGGGGICAAGGA ACAGGGTACCAA
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC G
CGTTCGGACAGG
ACCCTGGTCACCG EGTOGAGATCAA
GAGACICCCGGGACCICAGAGTCCGCCAC
GTACCAAGGTGG mo n TCTCCTCG A
ACCCGAAAGTGGIGGCGGA
AGATCAAATGA
______________________________________________ _ .
, be ' Co tc) 4...:1 c en cm t LH

C
U) A
A

Ln N) N) 17' A
r.) Table 18 w be Co be o ID N- SEQ SEQ VH-VL N- SEC/ SEQ Ft fusion C- 'SEQ SEQ VH-VL C. 5E4 _ tit ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VII NO: NO: VI NO: NO:
WI NO: NO: VL NO:
(VH1) (VU) (V113) (VL4) CTCGAGgacasaactescscaMAGTGOAGCC 2539 49 GAGGITCAGCIGGIG 46 GGTGGA 2542 50 GATATCCAGATG
knob- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACTTCTsitispeccatactTGCCCACC GAGICTGGCGGIGGC GGTGGC
ACCCAGICCCCG

AGCTCCCTGICC

CGTCASTCTICCTCTICCCCCCAAAACCCA GECTCACTCCGITTET
GCCTCTGTGGGC
TCCGITTGICCTGT TAGGGICACCAIC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGICACAIGCGTGGIGGIGGACGTGAGC CITCAACATCTLITATT
ATCACCTGCCGT
TCAACCTCTCITCT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGMAACTG CTICIATCCACTGGGT
GCCAGICAGICC
TATICTATGCACIG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGICCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGIA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGIGGICAGCGTCC GGITGCATATATTICT
CAACAGAPACCA
IGGAATGGGTTGC GCTCCGMGCTIC
TCACCGICCIGCACCAGGACTGGCT'GAAT TCTTATTAIGGCTATA
GGAAAAGCTCCE
µe=
cb ATATATTICTICIT TGATITACTCGGC
GGCAAGGAGIACAAGTGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTICTGATT
ATTATGGCTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTAIGCCGATA TCTGGAGTCCCTI
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AUCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG

GGAGICCCTICT
TTTCACTATAAGCG TAGCCGTICCGG
CGGGAGGAGAIGACCAAGA.ACCAGGTCA CCIACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTICACT
GCCTGIGGTGCATGGICAAGGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTICCGGG
AAACACAGCCIAC CIGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
ECAATGGGCAGCCGGAGAACAACTACAA TATIGTGLICGCGCTC
CTGACCATCAGC
GCTIAAGAGCTGA AAGACTICGCAAC
GACCACGCCTCCCGIGCTGGACTCCGACG ATTACTTCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC ITATTACTGICAG
GCTCCTICTTCCTGTACAGCMGCTCACCG TEGTGCTATEGACTAC
GAAGACTICGCA

TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTTATTACTGTC
CCCGGCTCCGGGT CICCGATCACGTI
CGICTICTCATGCTCCGTGATGCATGAGGC CTGGTCACCGICTCCT
AGCAATACTACT
CATTGGGGITTTG CGOACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACGT
ACTACTGEGGICA CAAGGTGGAGAT
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC
TCGGACAGGGTA
'I
AGGAACCCTGGIC CAAA
GAGACICCCOGGACCICAGAGICCGCCAC
CCMGGTGGAG n ACCGICTCCTCG
ACCCGAAAGTGGIGGCGGA
ATCAAATGA

C
be S...:' c lit cm =
t tee C
U) A
A

N) N) 17' i-a Table 1B
r.) be o ID N. SEQ SEQ 1/14411. N. SEQ SEQ
FetusIon C. SEQ SEQ VII-V1. C- SEQ
til ia fusion ID ED linker fusion ID ID fusion ID ID tinker fusion ID
VII NO: NO VI NO: NO:
VH NO: NO: VI NO:
(VIM 11/1.2) (VH3) (V) 56 CTCGAGgatasisetestaciAAAGTTGAGCC 2542 53 GAGGITCAGCTGGIG 46 GGTGGA 2539 hole- TGGAGTCTGGCGG GETGGC ACCCAGTCCCCGA
CAAATCTTCTgataagacccateetTGCCCACC GAGTCTGGCGGTCiGC GGTOGC

AGCTCCCTGTCC

CGTCAGTCTECCICTTCCCCCCAAAACCCA GGCTCACTCCGTTTET
GCCTCTGTGGGC
TCCGITTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGIGGTGGACGTGAGC CTICAACATCTCTTCTT
ATCACCTGCCGT
TCAACCTCTCTTCT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG ATTATATCCACTGGGT
GCCAGTCAGTCC
TATTCTATGCACTG CAGCGCTGTAGC
GTACGTEGACGGCGTGGAGGIGCATAATG G =CAC G CCCCG GG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAAC.AG
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCG GGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGICAGCGTCC GGTTGCATCTATTTAT
CAACAGAAACCA
TGGAATGGGTTGC GCTCCOMGCTIC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTICITAIGGCTATA
GGAAAAGCTCCG
c, ATATATTTCTTCTT TGATTTACTCGGC
GGCAAGGAGTAC.AAGTGCAAGGICTCCAA CTTCTTATGCCGATAG
AAGCTTCTGAIT

ATTATGGCTATACT ATCCAGCCTCTAC

TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT

AGOCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGIGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTTCT
TTTCACTATAAGCG TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AMCACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCG CCGTG GAGTGG GAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG

CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGOACTCCGACG TTCGTGGATCCAAAAA
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGICAG
GCTCCITCTTCCTCGTGAGC.MGCTCACCG gCGTACTTc i c i GGT
GAAGACTTCGCA
TATTATTGTGCTCG CAATGGTACTACG
TGGACAAGAGCAGGIGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTTATTACYGTC
CCCGGCTCCGGGr CTCCGATCACGTT
CGTCTTCTCATGCTCCGTGATGCATGAGGC GGGGTCAAGGAACCC
AGCAATACTC11.
CATTGGGGTTTTG CGGACAGGGTAC
TCTGCACAACCACTACAC6CAGAAGAGCC TGGICACCGTCYCCIt GGGGTCCGTTCA

TCTCCCTGTCTCCGSGTAAAAGCGGCAGC G
CGTTCGGACAGG
AGGAACCCTGGIC CAM
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTGG mo n ACCGTCTCCTCG
ACCCGAAAGTGGIGGCGGA
AGATCAAATGA
0 be CD
t-4 till LA
t C
0.) A
A

I
Ln IV

N) 17' ,--, Table la NJ

be C:1 NO
CI
ID N- SEQ SEQ VII-V1 N. SEQ ¨SEQ Fe fusion Cr SEQ SEQ VII-18. C- SEQ
tit ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO; NO: VI. NO: NO:
VII NO: NO: VI NO:
(VH1) (V12) (VHS) {VIA) 5048- 54)48 59 GAGGITCAGCTGG 46 GGTGGA 5048 60 GATATCCAGATG 48 CICGAGsacsassetcacantAAAGTGGAGCC 2539 49 GAGGITCAGCTGGTG 46 GGTGGA

knob- TGGAGTCTGGCGG GGTGGC = ACCCAGTCCCCGA
CAAAACTTCTgattagacccatactTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAAcTccTGGGGGGAc CTIGTGCAGCCAGGG AGT
AGCTCCCTUCC

CGTCAGTCTTCCTCTTCCCCCCAAAACCCA GGCTCACICCGTTIGT
GCCICIGIGGGC
TCCGTTTGICCIGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CaGIGCAGCTTCYGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGTGGTGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACATCTCTIAT GICAGTCCGTGTC

GCCAGTCAGTCC
TATTATATGCACTG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCIGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGICAGCGTCC GGTTGCATATATTICT
CAACAGAAACCA
TGGAATGGGTTGC GCTCCGAAGCTIC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTAITATGGCTATA
GGAAAAGCTCCG
µ2, ATCTA1TIC1TC1T TGATTTACTCGGC
EGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTATTATGCCGATAG

oo ATTATGGCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC

CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
. GCGTCAAGGGCCG CICGCTTCYCTGG
GAACCAATGGTOTTTGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTICT

CGGGAGGAGATGACCAAGAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTUGGTGCATGGICAAGGGCTICTAT CAGCITAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGICTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATOGGCAGCCGGAGAACAACTACAA TATTGTGCYCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACTICCCGTGGGC
AGTCTGCAGCCG

GAAGACTTCGCA
TATTATTGTGCTCG CAACATTACTCTG
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTIATTACTGIC
CTCTTGGTGGGCT TTTACGCTTCICT
CGTCTTCTCATGCTCCGTGATGCATGAGGC CTGGTCACCETCTCCT =
AGCAATACTACT
' TGGGCTTTTGACT GATCACGTTCGG
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCAOGT
ACTGGGGTCAAGG ACAGGGTACCAA
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC
TCGGACAGGGTA
AACCOGGICACC GGYGGAGATCAA
GAGACTcccGGGACCTCAGAGTCCGCCAC
CCAAGGTGGAG mo n GTCTCCTCG A
ACCCGAAAGTGGTGGCGCA
ATCAAATGA

Co t-4 5...:1 c In t Go C
U) A
A

Ln N) N) 17' A
r.) Table 113 w Cl be Co o ID N- SEQ .SEQ VII-VL N- SEQ SEQ. Fc fusion C= SEQ HQ VII-VI. C- SEQ
tit ia fusion PD ID linker fusion ID ID
fusion ID ID linker fusion ID
VH NO: NO: Vt. NO: NO;
VH NO: NO: VI. NO:
(VH1) (1/1.2) 0/113) (V1.4) 5048. 5048 59 GAGGTTCAGCTGG 46 GGTGGA S048 60 GATATCCAGATG 56 CTCGAGgsessaacteseseaAAAGITGAGCC 2542 53 GAGGITCAGCTGOTG 46 GGTGGA 2539 52 GATATCCAGATG
hole- TCGAGICTGGCGG GGTGGC ACCCAGTCCCCGA
CAMICTICTgatnaporataatTGCCCACC GAGICIGGCGGTGGC GGTGGC

2539. TCGCCIGGIGCAG AGT GCTCCCIGTCCGC

AGCTCCCIGTCC

CGTCAGTCTICCTCTICCCCCCAAAACCCA GGCTCACICCGTTTGI
GCCTCTGTGGGC
TCCGTTIGTCCTGT TAGGGTCACCATC

GATAGGGTCACC
GCAGCTICTGGCT ACCTGCCGTGCCA
AGGTCACATGCGIGGIGGIGGACGTGAGC CTTCAACATCTCTICIT
ATcACCTGCC5T
TCMCATCTCTTAT GTCAGTCCGTGIC
CACGAAGACCCTGAGGICAAMICAACTG ATTATATCCACTOGGT
GCCAGTCAGTCC
TATTATATGCACTG CAGCGCTGTAGC
GTACGTGGACGGCGIGGAGGIGCATAATG GCGTCAGGCCCCGGG
GTGICCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAMGCCGCGGGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
C,AACAGCACGTACCGT6TGGICAGCGTCC GOTTGCATCTATTTAT
CAACAGAAACCA
TGGAAIGGGITGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTICTTATGGCTATA
GGAMAGCTCCG
µ2, u:s ATCTATTTCTICTT TGATTTACTCGGC

AAGCTTCTGATT
ATTATGGCTCTACT ATCCAGCCICTAC
CAAAGCCCTCCCAGCCCCCATCGAGMAA CGTCAAGGGCCGTTTC
TACTCGGCATCC

CCATCTCCAMGCCAAAGGGC.AGCCCCGA ACTATAAGCGCAGAC
AGCCICTACTCI
GCGTCAAGGGCCG CICGCTICTCTGG
GAACCACAGGIGTACACCCTGCCCCCAAT ACATCC.AAAAACACAG
GGAGTCCCTTCT
TTTCACTATAAGCG TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAMTGAA
CGCTETCTGGT
C.AGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTICTAT CAGCTTMGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
g6SATPICACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
GCTIMGAGCTGA AAGACTTCGCMC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGEATCCAAAAA
AGTCTSCAGCCG
GGACACTGCCGTC TTATTACTGICAG

GAAGACTTCGCA

TGGACAAGAGCAGGTGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTIATTACTGIC
CICTTGGIGGGCT ITTACGCTICTCT
CGETTCTCATGCTCCGTGATGCATCAGGC GGGGICAAGGAACCC
AGCAATACTCTT
TGGGCMIGACT GATCACGTICGG
TCT6CACAACCACTACACGC.AGAAGAGCC TGGICACCGTCTCCTC
GGGGTCCGTTCA

TCTCCCTGICTCCGGGIAAAAGCGGCAGC G
CETTCGGACAGG
AACCCIGGTCACC GGTGGAGATCAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTGG mo n GTCTCCTCG A

AGATCAAATGA

be Co t-e S...:1 c en c.n t tee C
U) A
A

Ln N) N) 17' 1--, Table 18 r.) ...., ' No ct.
No co ID N. SEQ SEQ IWO!! II- SEQ - SEQ
Fcfusion C- SEQ SEQ VH-VL C- SEQ
rito ia fusion ID ID linker fusion ID ID
fusion ID ID linker fuslon ID
VH NO: NO: VL NO: NO:
VII NO: NO: VL NO;
(VHI) (VI-21 (VHS) (iL4) CTCGAGgacassactcaaciAAAGTGGAGCC 2539 49 GAGGTTCAGCTGGTG 46 GGTGGA 2542 knob- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACITCTgataigacccatactTGCCC.ACC GAGTCTGGCGGIGGC GGTGGC
ACCCAGTCCCCG
2539- TGGCCIGGIGCAG AoT GCrCCCTGTCCGC
GTGCCCAGCACCTGAAcTccT0000lsoAc CTGGTGCAGCC.AGGG AGT
AGCTCCCTGTCC

CGICAGTCTTCCTCTTCCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCTCTGTGGGC

AGGACACCCTCATGATCTCCCGGACCCCTG CCTGIGC.AGCTTCT56 GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGTGGIGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACatctettallott GTCAGTCCGTGIC
CACGAAGACCCTEIAGGTCA.45TTCAACT6 CTTCTATCCACTGGGT
GCCAOTCAGATCC
atatoCACTGGGTG CAGCGCTGTAGC
GIACGIGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
CGICAGGCCCOG CTGGTATCMCAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCC'TGGTAT
GTAAGGGCCTGGA AAACCAGGAAAA
CAACAGCACGTACCGIGTEGTCAGCGTCC GGTTGCATATAITTCT
CAACAGAAACCA
ATGGGTTGCATCT GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
iATTTATTCTICTTC TGAMACTCGGC
GGCAAGGAGTACAAGTGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTICTGAll TAGCTATACTIATI ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGMC
TACTCGGCATCC
AIGCCIATAGCGT TCTGGAGTCCC11' CCATCTCCAMGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT

GAACCAATGGTGITTGACCTGCCCCCATcc ACATCCAAAAACACAG
GGAGTCCCTTCT
ACTATAAGCGCAG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
ACACATCCAAAAA GACGGATTTCACT
GCCTUGGIGCATGGICAAGGGCTICTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGE
CACAGCCrACCTA CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCIAT
ACGGATTICACT
CAAATGAACAGCT GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGIGCTCGCGCTC
CTGACCATCAGC
TAAGAGCTGAGGA MGACTTCGCAAC

AGTCTGCAGCCG
CACTGCCGTCTATT TrA11ACI3Tr.A6 GAAGACTTCGCA
ATTGTGCTCGCTCI CAATCTGGTIGGT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTTATTACTGTC
TCTTACGCTIGGG GGGOTGITTCTCT

AGCAATACTACT
CTATTGACTACTG GATCACGTTCGG
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACGT
GGGTCMGGAACC ACAGGGTACCAA
TCTCCCTGICTCCGGGTAAAAGCGGCAGC
ICGGACAGGGIA
li CTGGICACCGTCT
GGTGGAGATCAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC CCAAGGTEGAG n CCTCG A
ACCCGAAAGTGGIGGCGGA
ATCAAATGA

No C:, No 5...:' c In Loo t coo C
U) A
A

N) N) 17' 1--, Table 16 N., w No C
No ID N- SEQ SEQ VH-1/1. N. SEQ SEQ Fc fusion C- SEQ SEQ VH-VI. C- SEQ
riti ia fuslon ID ID linker fuskon ID ID
fusion ID ID linker fusion ID
VII NO: NO; Vt NO: NO:
VII NO: NO: VI. NO:
(011) M2) 1V1151 (VIA) 56 CTCGAGgacaaaancacaraAAAeTTGAGCC 2542 53 GAGGITCAGCT6GTG 46 GGTGGA 2539 hole- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAATCTICTgataspeccataatTGCCCACC GAGTCTGGCGGTGGC GETEGC
ACCCAGTCCCCG
2539- TGGCCTGGTGCAG AGT .GCTCCCTGICCGC
GTGCCCAECACCTGAACTCCTGGGEGGAC CTGGTGCA6CCAGGG A31' CGTCAGIC/TCCFCITCCCCCCAAAACCCA GGCTtACTCCGTTTGT
GCCTLIGTGOGC
TCCGTTIGICCIGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCIG CCTGTGCAGCTICTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGIGGTGGACGTGAGC CTTCAACATCTCTICTT
ATCACCTGCCGT
TCAACatctcHattatt GTCAGTCCGTGIC
CACGAAGACCCTGAGGICAAGTICAACTG ATTATATCCACTGGGI
GCCAGTCAGTCC
ststrCACTGGGTG CAGCGCTGTAGC
GIACGTGGACGGCGIGGAGGIGCATAAIG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
CGTCACGCCCCGG CIGGTATCAACAG
CCAAGACAA.A.GCCGCGGGAGGAGCAGTA TAAGGGCCTG GAATG
GTAGCCTGGTAT
GTAAGGGCCTGGA AAACCAGGAAAA
CMCAGCACGTACCGTGTGGTCAGCGTCC GGTTGCATCIATTTAT
CAACAGAAACCA
I.L ATGGGTTGCATCT GCTCCGAAGC1TC
TCACCGTCCTOCACCAGGACTGGCTGAAT TCTICTTAIGGCTATA
GGAAAAGCTCCG
0 = ATTTATTCTTCTTC TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTCTTATGCCGATAG
AAGCTTCTGATT
=L
TAGCTATACTTATT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
ATGCCGATAGCGT TCTGGAGTCCCTT
CCATCTCCAMGCCAAAGGGCMCCCCGA ACTATAAGDGCAGAC
AGCCTCTACTCT

GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTTCT
ACTATAAGCGCAG TAGCCGTICCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAAATGAA
CGCTTCYCIGGT
ACACATCCAAAAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTICTAT CAGOTAAGAGCTGA
AGCCGMCGSG
CACAGCCTACCTA CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CAAATGAACAGCT GICTGC.AGCCGG
GCAATGGGCAGCCGGAGMCAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
TAAGAGCTGAGGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGIGGATCCAAAAA
AGICIICAGCCG
CACTGCCGTCTATT TTATTACTGTCAG
GCTCCTICTTCCTCGTGAGCAAGCTCACCG ACCGTACTTCTCTGGI
GAAGACTTCGCA
ATIGTECTCGCTCT CAATCTGGTTGGT
TGEACAAGAGCAGGIGGCAGCAGGGGAA TGGGCTATCGACTACT
ACTTATTACTGIC
TCTTACGCTTGGG GGGETGTITCTCT
CGICTICTCATGCTCCGTGATGCATGAGGC GGGGICAAGGAACCC
AGCAATACICTI
:
CTATTGACFACTG GAICACGTTCGE
TCMCACAACCACFACACGCAGAAGAGCC TGGICACCGICICCTC
GeGGICCGTTCA
GGGTCAAGGAACC ACAGGETACCAA
TCTCCCTGICTCCGGGTAAAAGCGGCAGC G
CGTTCGGACAGG
CIGGTCACCGTCT GGTGGAGATCAA
GAGACICCCGGGACCTCAGAGICCGCCAC
GTACCAAGGIGG mo n CCTCG A
ACCCGAAAGTGGTGGCGGA
AGATCAAATGA

_ _ No S...:1 o tio t.n t Go C
U) A
A

Ln N) N) 17' A
r.) Table 1B
w be C

ID N. SEQ SEQ VH-VL N- SEQ SEQ Fc fusion C- SEQ SEQ VIM C. SEQ
tit ia Sian ID ID linksr Mion ID ID
fusion ID ID linker fusion 10 vH NO: NO: VL NO: NO:
VH NO: NO: VI NO:
(VH1) (VU) IVH3) (VII.4) CTCGAGgacsaautescacoMAGTEGAGCC 2539 49 GAGGTTCAGCTGGTG 46 GGTGGA 2542 50 GATATCCAGATG
knob- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAMACTICTgataasacctatettTGCCCACC GAGTCTGGCGGTGGC GGIGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCIGGOGGGAC CTGGTGCAGCCAGGG AGT

CGTCAGTCTTCCICTTCCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCICTGTOGGC

AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCC.GTGCCA
AGGTCACATGCGIGGIGGTGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACATerCTTAT GTCAGTCCGTGIC

GCCAGTCAGTCC
TATTATATCCAVG CAGCGCTGTAGC
GTACOTGGACGGCGIGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGIGCGTCAGGCC CTGGTATC.AACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
STAGCCIGGTAT
CCCGGTAAGGGCC AAACCAGGAAAA
CAAMCACGTACCGTGTGGTCAGCGTCC GGTTGCATATATTTCT
CAACAGAAACCA
I.L TGGAATGGGTTGC GC1CCGAAGCTIC
TCACCGTCCTGCACCAGGACTGGCTGMT TCTTATTATGGCTATA
GGAAAAGCTCCG

th.4 ATCTATTTATCCTT TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTATTATGCCGATAG
MGCTICTGATT
CTTCTGGCTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGMC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGGIGITTGACCTGCCCCCATOC ACATCCAAMACACAG
GGAGTCCCTTCT
TITCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGA.ACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTr-ACT
GCCIGIGGIGCATGGICAAGGGCTTCTAT CAGCTTAAGAGCTGA

AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATITCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACTTCCCGTGGGC
AGICTGCAGCCG
GGACACTGCCGTC TTATTACTGTOW
GCTCCTTCTICCTGTACAGCAAGCTCACCG TGGTSCIATGGACTAC
GAAGACTTCGCA
TATTATTGTGCTOG CAATCTTACGCTG
TGGAC.AAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTTATTACTGIC
CTCTICITTCTACT CTTACCTGITCAC
CGTCTICTCATGCTCCGTGATGCATGAGGC CTGGTCACCGTCTCCT
AGCAATACTACT
GGGCTATGGACTA UTTCGGACAGGG
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGC.CGATCACGT
CTGGGGTCAAGGA TACCAAGGTGGA
TCTCCCTGICTCCGGGTAAAAGCGGCAGC
TCGGACAGGGTA
li ACCCTGGTCACCG GATCAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCAAGGIGGAG n TCTCCTCG
ACCCGAAAGTGGIGGCEGA
ATCAAATGA

_ be Co S...:1 c In tin t too C
U) A
A

Ln N) N) 17' A
r.) Table 1E1 w ' ba t ra --...
ra cot C
, ID N- SR) SEQ VH-VL N- SEQ SEQ Fc fusion C- ISEQ SEQ VH-VI. C- __ SEQ
it fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VII NO: NO; M. NO: NO:
VH NO; NO: VL NO:
NO (VU) IIV113) (ViA) CTCGAGpcsa is ctes ea caAAAGTTGAGCC 2542 58 -GAGGTICAGCTGGTG 46 GGTGGA- 2539 hole- TGGAGTCTGGCG6 GGTGGC ACCCASTCCCCGA
CAAATCTICTgata op ectataatTGCCCACC GAGTCTGGCOGIGGC GGTGGC
AC_CCAGTCCC.CG
2539. TGGCCTGGTGCAG AGT GaCCCTUCCGC
GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAG CCAGGG ACT
AGCTCCCTGTCC

CGTCAGTOTCCICTICCCCCCAAAACCCA GGCTCACTCC GiTTGT
GCCICTGIGGGC
TCCGTTTGTCCTGT TAGGGICACcATC

GATAGGGTCACC
GCAGCTTcTGGa ACCTGCCGTGCCA
AGGTCACATGCGTGGIGGTGGACGTGAGC CTICAACATCTCTICIT
ATCACCTGCCGT
TCAACATCTCTTAT GTCAGICCGTOTC

GCCAGTCAGTCC

GTACGTEGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG ' GTGICCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG

GTAGCCTGOTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC GGTTGCATCTAITTAT
CAACAGAAACCA
1¨i TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTCTTATGGCTATA
GGAAPAGCTCCG
ci ta ATCTATTTATCCTT TGATITACTCGGC
GGCAAGGAGTACAAGTECAAGGTCTCCAA CTTCTTATGCCGATAG
AAGCTTCTGATT
CTTCTGGCTATACT ATCCAGttTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGITTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATMGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGIGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCITCT
TTTCACTATAAGCG TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATt CAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGETGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTEGGAGA GGACACTGCCGTCTAT
ACGGATT1tACT
CTACAAATGAACA ETCTGCAGCCGG
GCMTGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGGATCCAAAAA
AGTCTGCAGCCG
GGACACTGCCGTC TTATIACTGICAG
GCTCCTTCTTCCTCGTGAGCAAGCTCACCG ACCGTACTICTCTGGT
GAAGACTICGCA
TATTATTGTGCTCG C.AATCTTACGCTG
TGGAC.AAGAGCAGGIGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTTATTACTGTC
CTCTTCTTICTACT CTTACCTGTTCAC
CGTCTXTCATECTCCGTGATGCATGAGGC GGGGTCAAGGAACCC

GGGCTATGGACIA GTTCGGACAGGG
TCTGCACAACCACTACACGCAGAAGAGCC TGGTCACCGICTCCTC
GGGGTCCGTTCA
CTGGGGICAAGGA TACCAAGGTGGA

CGTTCGGACAGG
ACCCTGGTCACCG GATC.AAA
GAGACTCCCGGGACCICAGAGTCCGCCAC
GTACCAAGGTG my n TCTCCTCG
ACCCGAAAGTGGTSGCGGA
AGATCAAATGA
¨
_______________________________________________________________________________ _______________________________________________________________________________ _______________ ba e t4 e a LA

to.) C
U) A
A

Ln N) N) 17' A.
r.) Table 18 w be =:::.
o ID N- SEQ SEQ VII-VL 11- SEQ 5E0 Fc fusion =C- SEQ SEO, VII-VL C- SEQ
tit ia fusion ID ID linker Sion ID ID
fusion ID ID linker fusion ID
VII NO: NO: VL NO; NO:
Vlil NO: NO: VL NO:
(VIII) (Vii) (VHS) (VW
_ 5080- 5080 65 GAGGITCAGCTGG 46 GGTGGA 5080. 55 GATATCCAGATG 48 CTCGAGgacesanttscacsAAAGTOSAGCC 2539 49 GAGGTTCAGCTGGTG 46 GGTGGA 2542 50 GATATCCAGATG
knob- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACITCTgataagaccanactTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AG?
AOCTCCCTGTCC

CGTCAUCTECTCTICCCCCCAAAACCCA GGCTCACTCCGTTTGT , GCCICTGTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATC7CCCGGACCCCIG CCTGTGCAGCTTcTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGitACATGCGTGGTGGTOGACGIGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACATCTCTTAT GTCASTCCGTETC
CACGAAGACCCTGAGGTCAAGTTCAACTG CTICTATCCACTGGGT
GCCAGTCAGTCC
TATTCTATGCACTG CAGCGCTGTAGC
GTACGIGGACGGCGIGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCAOSTACCGTGTGGICAGCGTCC GGTTGCATATATTTCT
CAACAGAAACCA
TGGAATGGOTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACIGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
Er ATCTATTTCITCTI TGATTTACKGGC
GGCAAGGAGTACAAGTGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTTCTGATT
ATTATAGCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTX
TACTCGGCATCC
TCTTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGGTGTTIGACCIGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTTCT
TTTCACTATAAGCG TAGCCGTTCCOG
COGGAGGAGATGACCAAGAACCAGGICA CCTACCTACAMTGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGAMCACT
GCCTEGGIGCATGGTCMGGGCTICTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACIGCCGTCTAT
ACGGATTTCACT

GCAATGGGCAGCCGSAGAACAACTACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACMGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACTTCCCGTGGGC
AGICTSCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTICTICCTGTACAGCMGCTCACCG TGGTGCTATGGACTAC
GAAGACTICGCA
TATTATTGTGCTCG CAACATTGGTCTT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TEGGGTCMGGAACC
ACTTATTACTGTC
CTTCTGGTACCCG ACCCGATCACGTT
CGICTICTCATGCTCCETGATGCATGAGGC CTGGICACCGTCTCCT
AGCAATACTACT
GGTATGGACTACT CGGACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACGT
GGGGTCAAGGAAC CAAGGTGGAGAT
TCTCCCIGICTCCGGGTAAAAGCGSCAGC
TCGGACAGGGTA
li CCTGGTCACCGTC CAM
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCAAGGTGGAG n TCCTCG
ACCCGAAAGTGGTGGCGGA
ATCAAATGA

. _ C:, c en cm t too C
U) A
A

Ln N) N) 17' 1--, Table 1B
r.) w C
be *
o t.Z.3 ID 141- SEQ SEQ VH-VL N- SEQ SEQ Pc fusion ¨ --C- SEQ SEQ VH-VI. C- SEQ
tit *
fusion ID ID linker fusion ID ID fusion PD ID linker fusion ID
VII NO: NO: VI NO: No;
VH NO: NO: VL Nth (VHS) (VU) Mel (VIA) CTCGAGgscaseacttatacaAAAGTTGAGCC 2542 53 46 GGTGGA 2539 52 GATATCCAGATG
hole- TGGAGTCMGCGG GGTGGC ACCCAGTCCCCGA
CAAATCTTCTgatassacccataatTGCCCACC GAGGUCAGCTGGTG GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC GAGTCTGGCGGTGGC ACT
AGCTCCCTOTCC

CGICAGICITCCTCTICCCCCCAAAACCCA CTGGTGCAGCCAGGG
GCCTCTGTGQGC
TCCGTTIOTCCTGT 'TAGGGTCACCATC
AGGACACCCTCATGATCTCCCEGACCCCTG GGCTCACYCCGTTTGT
GATAGGGTCACC
GCASCITCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGIGGTGGIGGACGTGAGC CCTGTOCAGCTICTGG
ATCACCTGCCGT
TCAACATCTCTTAT GICAGTCCOTGIC
CACGAAGACCCTGAGGICAAGTTCAACTO CITCAACATCTCTTCTT
GCCAGTCAGTCC
TATTCTATGCACTG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG ATTATATCCACTGGGT
GIGICCAGCGCT
GOIGCGICAGGCC CTGGTATCAACAG
CCAAGACAsAGCCGCGGOAGGAGCAGTA GCGTCAGGCOCCOGG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCOTGIGGICAGCGICC TAAGGGCCTGGAATG
CAACAGAAACCA
I¨L TGGAATGGGTTGC GCTCCOAAGCTIC
TCACCGTCCTGCACCAGGACTGGCTGAAT GGITGCATCTATTIAT
GOAAAAGCTCCG
o ATCTAUTCTICTT
TGATTTACTCGGC GGCAAGGAGTACAAGTGCAAGGICTCCAA
TaTCTTATGGCTATA AAGCTTCTGATT
ATTATAGCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CTICTTATGCCGATAG
TACTCGGCATCC
TCTTATGCCGATA TCTGGAGTCCCIT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA CGTCAAGGGCCGTTTC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GMCCACAGGTGTACACCCTGCCCCCAAT ACTATAAGCGCAGAC
GGAGTCCCITCT
TTICACIATAAGCG vkaccorrecso CCGGGAGCTGATGACCAGCAACCAGGTCA ACATCCAAAAACACAG
CGCTTCTCTGGT
CAGACACATCCAA GACGOATTTCACT
GCCIGAGCTGCGCCGTCAAAGC CTTCTAT CCTACCTACAAATGAA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA CAGCTTAAGAGCTGA
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA GGACACTGCCGTCTAT
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TATTGTGCTCGCACTG
AGTCTGCAGCCG

GCTCCUCTICCICGIGAGCAAGCTCACCG TTCGTGGATCCAAAAA
GAAGACTTCGCA
TATTATTGTGCTCG CAACATTGGICTT
TGGACAAGAGCAGGTGGCAGCAGGGGAA ACCGTACTTCTCTGGT
ACTTATTACTGTC
CTTCTGGTACCCG ACCCGATCACGTT
CGICTICICATGCTCCGTGATGCATGAGGC TGGGCTATGGACTACT
AGCAATACTCTT
GGTATGGACTACT CGGACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC GGGGICAAGGAACCC
GGGGICCGTICA
GGGGTCAAGGAAC CAAGGTGGAGAT
ICTCCCTGICTCCGGGTAAAAGCGGCAGC TGGTCACCGTCTCCTC
CGTTCGGACAGG
li CCTGGICACCGTC [AAA
GAGACTCCCEGGACCTCAGAGTCCGCCAC G
GTACCAAGGTGG n TCCTCG
ACCCGAAAGTGGTGGCGGA
AGATCAAATGA

be cl:, 5...:1 o t coo C
U) A
A

Ln N) N) 17' A
r.) Table 18 w I:I
be Cs ID N- SEQ SEQ VII-VL N- SEQ SEC( Fe fusion C- SEQ SEQ VH4fL C. SEQ
tit fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VII NO: NO: VL NO: NO:
WI NO: NO: VL NO:
(VH1) (VU) (V1131 (VIA) - , 5081. 5061 67 GAGGTTCAGCTGG 48 GGTGGA 5081. 68 GATATCCAGATG 48 CTCGAGgaraaaattcacacaAAAGTGGAGCC 2539 49 GAGGITCAGCTGGTG 46 GGTOGA 2542 59 GATATCCAGATG
knob- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACTICTgataagaucatactTGCCCACC GAGICTEGCGGIGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AGT
AGCTCCCTOTCC

CGTCAGTCTTCCTCTICCCCCCAAAACCCA GGCTCACTCCGITTGT
GCCTCTGTGGGC
TCCGTTIGTCCYGT TAGGGICACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GoacrrtroGc-r ACCTGCCGTGCCA
AGGICACATGCGTOGTOSTGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACCTCTCTTAT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG CTTCTATCCACTGGGT
GCCAGTCAGTCC
TATTATATGCACTG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGICCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGOCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC. AAACCAGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC GETTGCATATATTICT
CAACAGAAACCA
TEGAATGGGITGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
iATCTAMATTCTT TGATTTACTCGGC
GECAAGGAGTACAASTGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTTCTGATT
ATTCTGGCTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TArATGCCGATA TCTGGAGTCCCIT
CCATCTCCAAAGCC.AAAGGGCAGCCCCGA ACTATAAGCGCAGAC
ACCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGGTGITTGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTICT
TITCACTATAAGCE TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGAMCACT
GCCTGTGGTGCATGGICAAGGGCTTCTAT CAGCTTAAGAGCTGA
AGCCETTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATOGCCGIGGAGTGGGAGA GEACACTGCCGICTAT
ACGGATITCACT
CTACAAATGAACA GTCTGCAGCCGG
ECAATGCGCAGCCGGAGAACAACTACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACTTCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGICAG
GCTCCTTCITCCTGTACAGCAAGCTCACCG IGGTGCTATGGACTAC
GAAGACTTCGCA
TATTATTGTGCTCG CAAGGTGGTTGG
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGICAAGGAACC
ACTTATTACTGIC

CGTCTTCTCATGCTCCGTGATGCATGAGGC CTGGTCACCGTCTCCT
AGCAATACTACT
GGGLITTTGACTA TCGGACAGGGTA
TCTECACAACCACTACACGCAGAAGAGCC CG
GGCCGATCAOGT
CTGGGGTCAAGGA CCAAGGTGGAGA
TCTCCCTGICTCCGGSTAAAAGCGGCAGC
TCGGACAGGGTA
ACCCTCATCACCG TCAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCAAGGTGGAG mo n TCTCCICG
ACCCGAAAGTGGTGGCGGA
ATCA.AATGA

be CD
t4 S...:1 o CA
VI
t coo C
U) A
A

Ln N) N) 17' i--, Table 151 r.) ,I.,., be Co t-e o ID N- SEQ SEQ Vei-VI. N- SEQ SEQ Fc fusion C- SEQ SEQ VH-VI. C- 5E4 riti ia fusion ID ID linker fusion ID ID fusbn ID ID linker fusion ID
VH NO: NO: VI. NO: NO:
Vi4 NO: NCI: VI NO:
(VHS) (VU) (VH3) (1114) . .

CTCGAGgacaaaacteacacaAAAGTTGAGCC 2542 53 GAGGTTCAGCTGGTG 45 GGTGGA 2539 52 hole- TGGAGICIGGCGE GGTGGC ACCCAGTCCCCGA
CAAATCTICTgateagaccicetaatTGCCCACC GAGTCTGGCGGTGGC GOTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AGT
AGCTCCCTGTCC

GCCTCTGTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCACATGCGTGGTEGTGGACGTGAGC CTICAACATCTCTICTT
ATCACCTGCOGT
TCAACCTCTCTTAT GTCAGTCCGIGIC
CACGAAGACCCTGAGGICAAGTTCAACTG ATTATATCCACTEGGT
GCCAGTCAGTCC
TATTATATGCACTG CAGCGCIGTAGC
GTACGTGGACGGCGTGGAGGIGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT =
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCIGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGC.ACGTACCGTGIGGICAGCGTCC GG7TGCATCTAITTAT
rsoracomecA
I¨L TGGAATGGGTTGC 3CTCCGAAGCTIC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTICTTATGGCTATA
GGAAAAGCTCCG
= ATCTAMATTCTT TGArTACTCGGC

MGCTICTGATT
-.I
ATTCTGGCTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCICTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTTCT
MCACTATAAGCG TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA MTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCMCGCCGICAAAGGCTICTAT CAGCTTMGAGCTGA ..se¨
AGCCGTTCCGEG
AAAC.ACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT

ECAATGEGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG, CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
EACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGGATCCMAAA
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTICITCCICGTGAGCAAGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
7ATTA1TGTGCTcG CAAGGTGGTTGG
TGGACAAGAGCAGGIGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTTATTACTGTC
CTCTICITTCGCTT GGICCETTCACGT
CGICTICTCATGCTCCETGATGCATGAGGC GGGGTCAAGGAACCC
AGCAATACTCTT
GGGCTTTTGACTA TCGGACAGGGTA
TCTGCACAACCACTACACGCAGAAGAGCC TGGICACCGICTCCTC
GGGGTCCGTTCA

CGTTCGGACAGG
li ACCC7GGTCACCG TcAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTGG n TCTCCTCG
ACCCGAAAGTGGTGGCGGA
AGATCAAATGA

.
-Co t-e 5...:1 c In cm t tee C
U) A
A

Ln N) N) 17' i--=
Table 18 r.) w ei:l be =:::.
o L.Z.3 JD N- SEQ, 514 VH-VL fel- SEC( SEQ Fc fusion C. SEQ SEQ VII-VL C- SEQ
tit ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VII NO: NO: VI. NO; NO:
vH No: NO: VI NO:
(VH2) (V12) (VH3) (M) 2928- 292$ 69 GAGGTTCAGCTGG 46 GGTGGA 2928 70 GATATCCAGATG 48 CTCGAGg ctscacIAAAGTGGAGCC 2539 49 GAGGTTCAGCTGGTG 45 GGTGGA 2542 50 GATATCCAGATG
knob- TGGAGTCTGGCGE GGTGGC ACCCAGTCCCCGA
CAAAACTTCTgataageacataciTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACC/GAACTCCIGGOOGGAC CTGGTGCAGCCAGGG = AGT
AGCTCCCTGICC

CGTCAGICTICCTCTTCCCCCCA.AAACCCA GGCTCACTCCGITTGT
GCCTCTGTGGGC
TCCGTTTGTCCIGT TAGGGTCACCATC
AGGACACCCTCATGATCTCC.CGGACCCCTG CCTGTGCAGCTICTEG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGICACATGCGTGGTGGIGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTOCCGT
TCAACATCTCTTAT GTCAGICCGIGTC
CACGAAGACCCTGAGGTCAAGTICAACTG CTICTATCCACTGGGT
GCCAGTCAGICC
TCTICTATCCACTG CAGCGCTGTAGC
GTACGIGGACGGCGIGGAGGTGCATAATG GCGTCAGGCCCCOGG
GTGTCCAGCGCT
GUTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
COGGGTAAGGGCC AAACCAGGAAAA
C.AACAECACGTACCGTGTGGTCAGCGTCC 6611GCATATATTTCI
CAACAGAAACCA
TGGAATOGGTICC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCITATTATGGCTATA
GGAMAGCTCCG
iATCTATTTATCCTI TGATTIACTCGGC

AAGCTTCTGATT
CTTATAGCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGICCCIT
CCATCTCCAMGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTICTCTGG
GAACCAATGGIGTTTGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCUTCT
TTTCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGMCCAGGICA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCIGTGGIGCATGGICAAGGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCOTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGAMCACT
CTACAAATGAACA GICTGCAGCCGG
GCMTGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACITCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTTCTTCCTGTACAGCAAGCTCACCG TGGIGCTATGEACTAC
GMGACTTCGCA
TATTATIGTGCTCG CAAGCrntrAcT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGICAAGGAACC
ACTTATTACTGTC
CTACTACGCTATG ACCCGATCACGTT
CGTCTICTCATGCTCCGTGATGCATGAGGC CTGGICACCGICICCT
AGCAATACTACT
GACTACIGGGGit CGGACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACGT
AAGGAACCCTGET CAAGGTGGAGAT
TCTCCCTGTCICCEGGTAAAAGCGGCAGC
TCGGACAGGGTA
li CACCGTCTCCTCG CAM
GAGACTCCCGGGACCTCAGAGICCGCCAC
CCAAGGIGGAG n ' ACCCGAAAGIGGTGGCGGA
ATCAAATGA

be cl;
t-4 c tit cm t LH

C
U) A
A

Ln N) N) 17' A
r.) Table 113 w be C
t-a o L.Z.3 0 N- HQ SEQ alili-VL ii- sell SEQ Fc fusion C- SEQ SEQ VII-VL C- SEQ
roil ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VN NO: NO: VL NO: NO:
VII NO: NO: vL NO:
(Viii) WM
[V113) (VL4) 56 CTCGAGgacesaacitacacaMAGTTGAGCC 2542 53 GAGGITCAGCT6GTG ' 46 GGTGGA

bore- TGGAGTCTGGCGG GGTGGC ACCCAGTCCCCGA
CAAAICTICThatasgacccatastTGCCCACC GAGICTG6C6GTGGC GGTGGC
ACCCAGICCCCG
2539- TOGCCTGGIGCAG AG? GCTCCCTGTCCGC
GTGCCCAGCACCTGAACTCCTGGOGGGAC CIGGTGCAGCCAGGG AG?
AGCTCCCTGTCC

CGTCAGTCTTCaa7CCCCCCAAAACCCA GGCTCACTCCGTITGT
GCCTCTGIGGGC
TCCGTTTGICCTGT TAGGGTCACCATC
AGGACACCCTCATGATCfCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTICTGGCT ACCTGCCGTGCCA

ATCACCTGCCGT
TCAACATCTCTTAT GICAGTCCGTOTC
CACGAAGACCCTGAGGTCAAGTTCAACTG ATTATATCCACTGGGI
GCCAGTCAGTCC
TCTICTATCCACTE CAGCGCTGTAGC
GTACGIGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCEGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAMA
CAACAGCACGTACCGTGTGGTCAGCGTCC GGTTGCATCTATTTAT
CAACAGAAACCA
TOGAATOGGTTGC GCTCCGAAGCTTC
ICACCGICCIGCACCAGGACTGGCTGAAT TCTICTTAIGGCTATA
GGAAAAGCTCCG
iATCTATTTATCCTT TGATITACTCGCC
GGCAAGGAGTACAAGTECAAGGTCTCCAA CTTCTTATGCCGATAG
AAGCTITTGATT
CTTATAGCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCIT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCOCAGAC

GCGTCAAGGGCCG CTCGCTTCICTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTICT
TTTCACTATAAGCG TAGCCOITCCOG
CCGGGAGCTGATGACCAGC.AACCAGGTCA CCTACCTACAAATGM
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTMGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCG CCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
GCMTGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGGATCCAAAAA
AGICTECAGCCG
GGAC.ACTGCCGTC 17ATTACTGTCAG
GCTCCTICTICCICGIGAGCMGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG CAAKTTETACT
TGGACAAGAGCAGGIGGCAGCAGGGEAA TGGGCTATGGACTACT
ACTTATTACTGTC
CTACTACGCTATG ACCCGATCACGTT
CGTCTTCTCATGCTCCGTGATGCATGAGGC GGGGTCAAGGAACCC

GACTACTGOGGIC CGGACAGGGTAC
TCTGCACAACCACTACACGCAGMGAGCC TGGICACCGTOCCIC
GOGGICCGITCA
AAGGAACCCIGGT CAAGGIGGAGAT

CGTTCGGACAGG
li CACCGTCTCCTCG CAAA
GAGACTCCCGGGACCTC.AGAGICCGCCAC
GTACCAAGGTGG n ACCCGAAAGTGGTGGCGGA

AGATCAAATGA

be C:, No 5...:1 c tai t coo C
U) A
A

Ln N) N) 17' A
r.) Table 18 w be C
a ID N- SKI SECI VH4L II- SEQ SKI Fc fusion C- SKI SEC/ VINVL C- SRI
tit ia fuslan ID ID linker fusion ID ID fusion ID ID linker fusion II) VII No: NO: VL NO: NO:
V1.1 NO: NO: VL NO:
WEI IVL2) (VH) (VIA) CTCGAGgacaseactcsocaAAAGTGGAGCC 2450 71 GAGGTTCAGCTGGTG 45 GONNA 2459 72 GATATCCAGATG
knob- TGGASICTGGCGG GGTGGC ACCCAGTCCCCGA
CAMACTICTgoteegacccatactTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGOGGGGAC CIGGIGCAGCCAGGG ACT
AGCTCCCTGICC

CGICAGTCUCCICTTCCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCTCTGTGGGC
TCCGITTGTOCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGIGCAGCTTCTOG
GAIAGGG/CACC
GCAGCTTCTGGCT ACCTOCCGTSCCA

ATCACCTGCCGT
TCAACATCGGTTCI GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG CTICTATACACTGGET
GCCAGTCAGTCC
TCTTCTATCCACTG , CAGCGCTGIAGC
GIACGIGGACGGCGTGGAGGIECATAATG GCGICAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGATAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT

CAACAGCACGTACCGTGTGGICAGCGTCC GGTTGCATCTATTICT
CAACAGMACCA
1¨L TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACIGGCTGMT TCTICTTAIGGCTATA
GGAAAAGCTCCG

e ATCTArTATTCTG TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTATTATGCCGATAG
AAGCTTCTGATT

CITTTGCCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGICAAGGGCCGTTIC
TACTCGGCATCC
TCTTATGCCGATA TCTGGAGTCCCIT
CCATCTCCAAAGCCAAAGGGCAGCCCOGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGICAAGGGCCG CICGCTTCICTGG
GAACCAATGGTGTITGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTDCDTICT
TTTCACTATAAGCG TAGCCGTTCCGG

CGCTTCICIGGT
CAGACACATCCM GACGGATTTCACT
GCCTGTGGTGCATGGICAAGGGCTICTAT C,AGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GeACACTGCCGTCIAT
ACGGATTICACT
CTACAAATGAACA GTCTGCAGCCGG
GCMTGGGCAGCCGGAGAACAACTACAA TATIGTGCTCGCGGIG
CTGACCATCAGC
GCTTAAGAGCTGA MGACTICECAAC
GACCACGCCTCCCGTGCTGGACTCCGACG GITICTGGTGTTTCTCA
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGICAG

GMGACTTCGCA
TATTATTGTGCTCG CAAGGTGMACC
TGGACMGAGCCGCTGGCAGCAGGGGAA TACTCTIGGIGGGCTT
ACTTATTACTGTC
CTACCATTTCCCGT IGTICACGTICGG
CGICTICTCATGCTCCGTGATGCATGAGGC IGGACTACTGGGGTC
AGCAAGCTTCTT
TCGGITTTGCITTG ACAGGGTACCAA
TCTGCACAACCACTACACGCAGAAGAGCC MGGAACCCIGGICA
ACGCTCCGATCA
GACIACTGGGGTC GGTGGAGATCM
TCTCCCTUCTCCGGGTMAAGCGGCAGC ' CCGTCTCCTCG
CGTTCGGACAGG
li AAG GAACCCIGGT A
GAGACTCCCGGGACCTCAGAGICCGCCAC
GTACCMGGTGG n CaCCGICTUTCG
ACCCGAAAGTGGIGGCGGA
AGATCAAA

be No 5-1:1 CA
t ca C
U) A
A

Ln N) N) 17' A
r.) Table 1B
w Ci No Co No co t.Z.3 ID N- SEQ SEQ VII-V1 N- SEQ SEQ Ft fusion C- SEQ SEQ VH-VL C. SEQ
rito ia fusion ID ID tinker fusion ID ID
fusion ID ID linker fusion ID
VII NO: NO: VL NO: NO:
VH NO: NO: In. NO:
(VII1) (VL2) (VHS) (VIA) CTCGAGisscsaasctroocaAAAGTTGAGCC 2459 73 GAGGITCAGCTGGIG 46 GGTGGA 2460 74 GATATCCAGATG
hole- IGGAGICIGCCGG GGTGGC ACCCAGTCCCCGA
CAAATMCTiptessioxstsatTGCCCACC GAGTCTGGCGGTGGC GGTIGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCIGOGOGGAC CTGOTGCAGCCAGGG AG?
AGCTCCCTUCC

TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTICTGGCT ACCTGCCGTGCCA
AGGICACATGCGTGGIGGIGGACGTGAGC CTICAACL:Li t.i i All ATCACCTGCCGT
TCAACATCGGITCT GTCAGTCCGTGTC
CACGAAGACCCTGAGGICAAGTICAACTG ATTATATGCACTGGGT
GCCAGICAGTCC
TCTTCTATCCACTG CAGCGCTGTAGC
GTACGTGGACGGCCITGGAGGTGCATAATG GCGTCAGGCCCCOGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCEGGAGGAGCAGTA TAAGGGCaGGAATG
GTAGCCTGGTAT
CCGSGTAAGGGCC AAACC.AGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC GGTTGCATCTATTTAT
CAACAGAAACCA
1¨i TGGAATGGGTTGC GCTCCGAAGCTIC
ICACCGICCTGCACCAGGACTGGCTGAAT TaTCTIATGGCTATA
GGAAAAGCTC.CG
1¨i 1¨i ATCTATTIATIICTG TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGGICTCCAA CTTATTATOCCGATAG
AAGCTTCTGATT
CTTTTGCCTCTACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TCTTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGICAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGTGTACACCCIGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTTCT
MCACTATAAGCE TAGCCGTTCCGG
CCGCGAGCTGATGACCAGCAACCAGGICA CCIACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCM GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGIGGGAGA GGACACTGCOGICTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCTGGT
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG CTCATGITTCTGGICA
AGTCTGC.AGCCG
GGACACTGCCGTC TIATTACIGICAG
GaCCTICTICCTCGTGAGCAAGCTCACCG TTACTCTGGTATGGAC
GAAGACTTCGCA
TATTATTGTGCTOG CAAGGTGTTTACC
TGGACAAGAGCAGGIGGCAGCAGGGGAA TACTGGGGICAAGGA
ACTTATTACIGTC
CTACCATTTCCCGT TGITCACGTTCGG

AGCAATCTTCTT
TCGGITTTGCTTIG ACAGGGTACCAA
TCTGCACAACCACTACACGCAGAAGAGCC CCTCG
ATTCTCTGATCA
GACTACTGGGGTC GGTGGAGATCAA
TCTCCCTGICTCCGGGIAAAAGCGGCAGC
CGTTCGGACAGG
li AAGGAACCCIGGT A
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTGG n CACCGTCTCCTCG
ACCCGAAAGTGGTGGCGGA
AGATCAAA

_..., No C:, No 5...:1 c en Loo t coo C
U) A
A

Ln N) N) 17' A
r.) Table 18 w be C
a ID N- HQ SEQ VH-111. il- HQ SEQ Fc fusion C- SEQ SEQ VIEVL C- SEQ
til ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NO: VL NO: NO:
VN NO: NO: VI NO:
(Will IVL2) (V/43) mil) 48 CTCGAGgacasaactrscscaAAAGTGGAGCC 2539 49 knob- TGGACCIGGCGG GGTGGC ACCCAGTCCCCGA
CAAAACITCTgataagacccatactTGCCCACC GAGTCTGGCGGTGGC GGIGGC
ACCCAGTCCCCG
. 2539- TGGCCTGGTGCAG AGT GCTCCCTGTCCGC
GTGCCCAGCACCTGAACTCCTGOGGGGAC CTGGIGCAGCCAGGG AGT
AGCTCCCTGTCC

CGTCAGTCITCCTCTTCCCCCCAAAACCCA GGCTCACTCCGITTGT
GCCTCTGTGGGC
TCCGTTTGICCTGT TAGGGTCACCATC

GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGTCAcATGCGTGGTGGTGGACGTGAGC CTICAACATCTCTTATT
ATCACCTGCOGT
TCAACATCTATTAT GTCAGTCCGTGIC
CACGAAGACCCTGAGGICAAGTICAACTG CTICIATCCACTGGGT
GCCAGTCAGTCC
TCTICIATCCACTG CAGCGCTGTAGC
GTACGIGGACGGCGTGGAGGIGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACMAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCIGGTAT
COGGGTAAGGGCC AMCCAGGAAAA
CAACAGCACGTACCGIGTGGICAGCGTCC GGTTGCATATATTTCT
CAACAGAAACCA
I.L TGGAATGGGTTGC GCTCCGAAGCITt TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG

EGCAAGGAGTACAAGTGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTICTGATT
bl ATTATGGCTATACT ATCCAGCCTCTAC
CAAJkGCCCTCCCAGCCCCCATCGAGAAAA CGTCAJkOGGCCGITTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCTT
CCATCTCCAMGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCICTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGOTGITTGACCTGCCCCC.ATCC ACATCCAAAAACACAG
GGAGICCCTICT
TTTCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGICA CCTACCTACAAATGAA
CGCTICTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGIGGTGCATGGICAAGGGCTTCTAT CAOCITAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGIGGGACA OGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
RAATGGGCAGCCGGAGAACAACTACAA TATTGTECTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCIGGACTCCGACG ATTACTTCCCGTGGGC
AGTCTSCAGCCG
GGACACTGCCGTC TTATTACTGICAG
GCTCCTTCTICCTGTACAGCAAGLICACCG TGGTGCTATGGACTAC
GAAGACTTCGCA
TATTATIGTGCTCG CAATCTTACTEGC
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCMGGAACC
ACTTATTACTGTC
CTACTACCATTACG ATTCTTACCTGAT
CGTCTICTCATGCTCCGTGATGCATGAGGC CTGGTCACCGTCTCCT
AGCAATACTACT
GITTGGACTACTG CACGTTCGGACA
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACGT
GGGTCAAGGAACC EGGTACCAAGGT
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC
TCGGACAGGGTA
li CTGGTCACCGTCT GGAGATCAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCMGGTGGAG n CCTCG
ACCCGAAAGTGGTGGCGGA
ATCAAATGA

, be CD
t4 5-1:1 c tit t coo C
U) A
A

Ln N) N) 17' 1--, Table 113 r.) w be C.

L.Z.3 13 N- SEQ SEQ 11,114-VL N- SEQ SEQ Fe fusion C.- SEQ SEQ VH-V1. C- SEQ
rito ia fusion ID ID linker fusion ID ID Sullon ID ID linker fusion ID
VII NO: NO: VI. NO: NO:
VII NO: NO: NM NO:
(V141) (VL2) OfG31 (V1.4) 2890- 2890 84 GAGGITCAGCTGG 4Ã GGTGGA 2890 85 GATATCCAGATG 56 CTCGAGgacaaasctescacaAAAGTIGAGCC 2542 53 hole- TGGAGICTGGCG6 GGTGGC ACCCAGTCCCCGA
CAAATCTTCTsstsaisceeataatTGCCCACC GAGTCTGGCGGTGGC GGTGGC
ACCCAGTCCCCG

GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGIGCAGCCAGGG AGT
AGCTCCCTGTCC

CGICAGICTICCTCTICCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCICIGTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCTGCCGTGCCA
AGGICACATGCGTGGIGGTGGACGTGAGC CTICAACATCTCTICIT
ATCACCTGCCGT
TCAACATCTATTAT GICAGICCGTOTC
CACGAAGACCCTGAGGTCAAGITCAACTG ATTATATCCACTGGGT
GCCAGTCAGTCC
TCTTCTATCCACTG CAGCGCTGTAGC

GTGTCCAGCGCT
GGIGCGICAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGGGAGGAGC.AGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCEGGTAAGGOCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC carrGemercrrar CAACAGAAACCA
1¨i TGGAATGGGITGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTCTTATGGCTATA
GGAAAAGCTCCG
1¨i ATCTAITTATCCTT TGATTTACTC6Gt CGCAAGGAGTACAAGTGCAAGGICICCAA CTTCTTATGCCGATAG
AAGCTTCTGATT

ATTATGGCTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGMC
TACTCGGCATCC
TATTATGCCGATA TCTOGAGTCCCIT
CCATCTCCAAAGCCAAAGGGCAGCCCCGA ACTATAAMICAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGICCCTICT
MCACTATAAGCE TAGCCGTTCCGG
CCGGGAGCTGATGACCAGCAACCAGGTCA :CCTACCTACAAATGAA
CGCTTCCTEGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCOGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGAMCACT
CTACAAATGAACA GTCTGCAGCCGG
ECAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CTGACCATCAGC
GCTTAAGAECTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TICGTGGATCCAAAAA
AGICTUCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTTCTTCCICGTGAGCAAGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG CAATCTTACTGGC
TGGACAAGAGCAGGTOGCAGCAGGGGAA IGGGCIATGGACIACT
ACTTATTACTGTC
CTACTACCATTACG ATTCTTACCTGAT
CGTCTICTCATGCTCCGTGATGCATGAGGC GGGGTCAAGGAACCC
AGCAATACCTT
GTTTGGACTACTG CACGTTCGGACA
TCTG CAC AAC CACTACAC GCAGAAGAG C C TGGTCACCGTCTCCTC
GGEGTCCGTICA
GGETCAAGGAACC GGGTACCAAGGT
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC G
CGTTCGGACAGG
CTGGICACCGICT GGAGATCAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTEG mo n CCTCG
ACCCGAAAGTGGTGGCGGA
AGATCAAATGA

C:, No 5-1:1 c in in t Go C
U) A
A

Ln N) N) 17' A
r.) Table 1.8 w ' No C
No co , ID N= SEQ SEQ Viit-VI. el- SEQ HQ Pc fusion C- SEQ ¨SEQ -VH.101. ¨C- SEQ
ia fusion ID ID linker fusion ID SD fusion ID ID linker fusion ID
VH NO: NO: VL NO: NO:
VH NO: NO: VL NO:
(VH1) (V12) me) (11o) 48 CTCGAGeataalacteatataAAAGTGGAGCC 2539 knob- TGGAGICTGGCGG GGTGGC 'ACCCAGTCCCCEIA
CAAAACTICTgataagaccestactIGCCCACC = GAGICTEGCGGIGGC GGTEGC
ACCCAGTCCCCG
2S39. TEGCCIGGTGCAG AGT GCTCCCTGTCCGC
GTGCCCACCACCTGAACTCCTOGGEGGAC CTGGTGCMICCAGGG AGT
AGCTCCCTGTCC
2542 CCAÃGGGGCTCAC CTCTGTGGGCGA
CGTCAGETTCCTCTICCCCCCAAPACCCA GGCTCACTCCGTTIGT
GCCTCTGTGGGC
TCCGTTIGTCCTET TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTGTGCAGCTICTGG
GATAGGGTCACC
GCAGCTICIGGCT ACCTGCCGTGCCA
AGGICACATGCGIGGIGGIGGACGTGAGC CITCAACATCTCTTATT
ATCACCTGCCGT
TCAACATCTOICT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG CITCTATCCACTGGGT
GCCAGTCAGTCC
TCTICTATGCACIG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG GCGMAGGCCCCGGG
GTGTCCAGCGCT
GGIGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCIGGAATG
GTAGCCIGGIAT
' CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC GGITGCATATATTICT
CAACAGAPACCA
I¨L TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
1¨L
.ia. ATCIAMATICIT TGAMACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTATTATGCCGATAG
AAGCTTCTGATT
ATTATGGCTCTACT ATCCACCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGMC
TACTCGGCATCC
TATTATGCCGATA MIGGAGTCCCTT
CCATCMCAAAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT

GAACCAATGGTGITTGACCTOCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCITCT
TTICACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACC.AAGAACCAGGTCA CCTACC7ACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGTGGTGCATGGTCAAGGGCTTCTAT C.AGCTTPAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGIGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACrACAA TATTGTGCTCGCGCTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ATTACTTCCCGTGGGC
AGTCTGCAGCCG
GGACACTGCCGTC ITATTACTGICAG
GCTCCITCTTCCTGTACAGCAAGCTCACCG TGGTGCTATGGACTAC
GAAGACTTCGCA
TATTATTGTGCTCG CAACCGGGTTCTT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTTATTACTGIC
CTGGTACCGTAIG GGTACTICCCGCC
CGTCTICTCAIGCTCCGTGATGCATGAGGC CTGGICACCGTCTCCT
AGCAATACTACT
GACTACTGGGGIC GATCACGTTCGG
TCTGCACAACCACMCACGCAGAAGAGCC CG
GGCCGATCACGT
AAGGAACCCTOGT ACAGGGTACCAA
TCTCCCTETCTCOGGGTAAAAGCGGCAGC
TCGGACAGGGTA
CACCGTCTCCTCG GGTGGAGATCAA
GAGACTCCCGGGACCTCAGAGTCCECCAC
CCAAGGTGGAG mo n A
ACCCGAAAGTGGIGGCGGA
ATCAAATGA

C:, No S...:' co en c.ro t roo C
U) A
A

Ln N) N) 17' 1--, Table 18 ,.) ,,., be Cs t=-.) cs t.Z.3 IC N. SRO 5112 VH-Vi. 141- SEQ SEQ
Winton C. Eta - SEQ VH-VL C- SEQ
tit ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NO: VI NO: NO:
VH NO: NO: VI NO:
(VHS) (VI.2) (VH3) (VI.4) 12735- 22735 86 GAGGTTCAGCTGG 46, GGTGGA 12735 87 GATATCCAGATG
56 'CITGAGpciasictcacacaAAAGTTGAGCC 2542 53 GAGGTICAGCTGGIG 46 hole- TGGAGICIGGCGG G(3TGGC ACCCAGICCCCGA
CAAATCTICTgetaapccestaatTGCCCACC GAGTLIGGCGGTGGC GGIGGC

2539. TGGCCTGGTGCAG AGT GCTCCCTOTCCGC
GTGCCCAGCACCTGAACTCCTGGGGGGAC CIGGIGCAGCCAGGG AU
AGCTCCCTGTCC

CGTCAGICTICCTCTTCCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCTCTGTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGMCTCCCGGACCCCTG CCTGTGCAGCTTCTGG
= GATAGGGTCACC
Graecrrasea ACCTGCCGTGCCA

ATCACCTGCCGT
' TCA.ACATCTCTICT GICAGTCCGTOTC

GCCAGTCAGTCC

GTACGTGEACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GTGTCCAGCGCT
GGTGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGGGAGGAGCAGTA TAAGGGCCIGGAATG
GTAGCCTGGTAT
COGGGTAAGGGCC AAACCAGIAAAA
CAACAGCACGTACCGTGTGGTCAGCGTCC GGTTGCATCTATTTAT
CAACAGAAACCA
I¨L TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAM TCTICITATGGCTATA
GGAAAAGCTCCG
1¨L ATCTAMATTCTT TGATTTACTCGIC

AAGCTICTGATT
tio ATTATGGCTCTACT ATCCAGCCTCTAC
CAMGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
. TACTCGGCATCC
TArATGCC6ATA TCTGGAGTCCCTT
CCATUCCMAGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
gracrAcia GCGTCAAGOGCCG CTCGCTXTCTGG
GAACCACAGGTGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
OGAGICCCTICT
MCACTATAAGCG TAGCCGTTCCGG
CCGGGAGCTGATEACCAGCMCCAGGICA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGAGCTGCGCCGTCAAAGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCOGTGGAGTGGISAGA GGACACTGCCGTCTAT
ACGGAMCACT
CTACAAATGAACA GTCTGCAGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATIIGTGCICGCACTG
CTGACCATCAGC
GCTTAAGAGCTGA AAGACITCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGGATCCAAAAA
AGICTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
ECTCCTTCTICCTCGTGAGCAAGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG CAACCGGGITCTT
TGGACAAGAGCAGGTGGCAGCAGGGGAA TGGGCTATGGACTACT
ACTTATTACTGTC
CTGGTACGGTATG GGTACTTCCCGCC
CGICTICTCATGCTCCGTGATGCATGAGGC GGGGICAAGGAACCC
AGCAATACTCTT
GACTACTGGGGTC GATCACGTTCGG

GGGGICCGTICA
MGGAACCCTGGT ACAGGGTACCAA
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC G
CGTTCGGACAGG
CACCGTCTCCTCG GGTGGAGATCAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGTGG mo n A
ACCCGAAAGTGGIGGCGGA
AGATCAMTGA

C
t=-.) 5-!.!
c .
en Ln t tee ' .

C
U) A
A

Ln N) 0 .
N) 17' A
r.) Table 1E1 w be C
t..) o i....3 ID N- SEQ SEQ VH-VI. N. SEQ SEQ Fe fusion C- SEQ SEQ VH-VL C- SEQ
ro it ia fusion ID :ID linker (unior: ID ID fusion ID ID
linker fusion iD .
VH NO: NO: VI. NO: NO:
VH NO: NO:. VL NO:
(V1.11) IVL2) IVH3) (VL4) CTCGAGgscaaalettscansAAAGTTGAGCC 2542 53 GAGGTTCAGCTGGIG 46 GGTGGA 2539 52 GATATCCAGATG
hole- TGGAGTCTGGCGG GGTGGC ACCCAGICCCCGA
CAAATCTTCTgatnagacccataatIGCCCACC GAGTCTGGCGGIGGC GGTGGC
ACCCAGTCCCCG

GTGCCC.AGCACCTGAACTCCTGGGiGGAC CTGGIGCAGCCAGGG AGT
AGCTCCCTGTCC

CGTCAGICTICCTCTICCCCCCAAAACCCA GGCTCACTCCGMET
GCCTCTGTGGGC
TCCGITTGICCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCIGTGCAGCTICTGG
GATAGGGTCACC
GCAGCTTCTGGCT ACCMCCGTGCCA
AGGTCACATGCGTGGTGGIGGACGTGAGC CTICAACATCTC-nYTT
ATCACCTGCCGT
TCAACICCTCTITT GTCAGTCCGTGTC
CACGAAGACCCTGAGGTCAAGTTCAACTG ATTATATCCACTGGGT
GCCAGTCAGTCC
TATTrTATGCACTG CAGCGCTGTAGC
GTACGTEGACGGCGIGGAGGTGCATAATG GCGTCAGGCCCCGGG
GIGTCCAGCGCT
GGTGCGTCAGGCC CIGGTATCAACAG

GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACETACCGTGIGGTCAGCGTCC GGTTGCATCTATTTAT
CAACAGAAACCA
I.: TGGAATGGGTTGC ICTCCGAAGCTIC
TCACCGTCCTGCACC.AGGACTGGCTGAAT TCITCTTATGGCTATA
GGAAA.AGCTCCG
1¨:
en AACTGITTATCCTT TGATTTACTCGGC
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CTTCTTATGCCGATAG
AAGCTTCTGATT
ATCTTGACTATACT ATCCAGCCTCTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTATGCCGATA TCTGGAGTCCCIT
CCATCTCCAMGCCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTICICIGG
GAACCACAGGIGTACACCCTGCCCCCAAT ACATCCAAAAACACAG
GGAGTCCCTTCT
ITICACTATAAGCG TAGCCGTTCCGG
CCOGGAGCTGATGACCAGCAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACAICCAA GACGGAMCACT
GCCTGAGCTGCGCCGTCAAAGG CTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AMCACAGCCTAC CTGACCATCASCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GICTGOLGCCGG
GCAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCACTG
CIGACCATCAGC
GCTTAAGAECTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG TTCGTGGATCCAMAA
AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGICAG
GCTCCTTCTTCCICGTGAGCMGCTCACCG ACCGTACTTCTCTGGT
GAAGACTTCGCA
TATTATTGTGCTCG CAATCTICTTATT
TGGACAAGAGCAGGIGGCAGCAGEGGAA TeGGCTATGGACTACT

COCGTrrecGGGT CTCTGATCACGTT
CGTCTICTCATGCTCCGTGATGCATGAGGC GGGGTC.AAGGAACCC
AGCAATACICTT
TCTTACCATCCTAT CGGACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC IGGTCACCGICTCCTC
GGGGTCCGTTCA
GGACTACIGGGGT CMEGTGGAGAT
TCTCCCTGICTCCGGGIAAAAGCGGCAGC a CGTTCGGACAGG
CAAGGAACCCTGG CAM
GAGACTCCCGGGACCTCAGAGTCCGCCAC
GTACCAAGGIGG mo n TCACCGTCTCCTCG
ACCCGAAAGTGGIGGCGGA
AGAICAAATGA

_ be C:, t..) 5...:' c en cm t La C
U) A
A

N) N) 17' 1--, Table 211 N., w be C.
C
ID N- SEQ SEQ VII-VL 141- SEQ SEQ Ft fusion C- SEQ SEQ VIM C- SEQ
riti ia fusion ID ID linker fusion ID ID fusion ID ID linker fusion ID
VH NO: NO: VI. NO; NO:
Vii NO: NO: VI. NO:
(VH1} MP
(V143) (VIA , CTCGAGgicisaactcacaciAAAGTGGAGCC 2539 49 GAGGTTC.AGCTGGTG 46 GGTIGA' 2542 SO
GATATCCAGATG
knob- TGGAGICTGECGG GGTGGC ACCCAGTCCCCGA
CAAAACTTCTgataagacccatactTGCCCACC GAGTCYGGCGGIGGC GGTGGC
ACCCAGTCCCCG
.2539- TGGCCTGGTGCAG AGT GCTCCCTGTCCGC
GTGCCCAGCACCTGAACTCCTGGGGGGAC CTGGTGCAGCCAGGG AGT
AGCTCCCTGTCC

CGTCAGICITCCTCTICCCCCCAAAACCCA GGCTCACTCCGTTTGT
GCCTCTOTGGGC
TCCGTTTGTCCTGT TAGGGTCACCATC
AGGACACCCTCATGATCTCCCGGACCCCTG CCTETGCAGCTICTGG
GATAGGGTCACC
GCAGCTICTGGCT ACCTGCCGIGCCA
AGGTCACATGCGTGGTGGIGGACGTGAGC CTTCAACATCTCTTATT
ATCACCTGCCGT
TCAACTCCTCTTTT GICAGTCCGTOTC
CACGAAGACCCTGAGGICAAGTICAACTG CTTCTATCCACTGGGT
GCCAGICAGTCC
TATTTTATGCACTG CAGCGCTGTAGC
GTACGTGGACGGCGTGGAGGTGCATAATG GCGTCAGGCCCCGGG
GIGTCCAGCGCT
GerGCGTCAGGCC CTGGTATCAACAG
CCAAGACAAAGCCGCGCGAGGAGCAGTA TAAGGGCCTGGAATG
GTAGCCTGGTAT
CCGGGTAAGGGCC AAACCAGGAAAA
CAACAGCACGTACCGTGIGGTCMCGTCC GGTTGCATATATITCT
CAACAGAAACCA
I.L TGGAATGGGTTGC GCTCCGAAGCTTC
TCACCGTCCTGCACCAGGACTGGCTGAAT TCTTATTATGGCTATA
GGAAAAGCTCCG
1¨L AACTGTTIATCCIT TGATITACTCGGC
GGCAAGGAGTACAAGIGCAAGGICTCCAA CTTATTATGCCGATAG
AAGCTICTGATT
=-.1 ATCTTGACTATACT ATCOACCIZTAC
CAAAGCCCTCCCAGCCCCCATCGAGAAAA CGTCAAGGGCCGTTTC
TACTCGGCATCC
TATTATGCCGATA TCTOGAGTCCCIT
CCATCTCCAMECCAAAGGGCAGCCCCGA ACTATAAGCGCAGAC
AGCCTCTACTCT
GCGTCAAGGGCCG CTCGCTTCTCTGG
GAACCAATGGTGTTTGACCTGCCCCCATCC ACATCCAAAAACACAG
GGAGTCCCTTCT
TITCACTATAAGCG TAGCCGTTCCGG
CGGGAGGAGATGACCAAGAACCAGGTCA CCTACCTACAAATGAA
CGCTTCTCTGGT
CAGACACATCCAA GACGGATTTCACT
GCCTGTGGTGCATGGICAAGGGCTTCTAT CAGCTTAAGAGCTGA
AGCCGTTCCGGG
AAACACAGCCTAC CTGACCATCAGCA
CCCAGCGACATCGCCGTGGAGTGGGAGA GGACACTGCCGTCTAT
ACGGATTTCACT
CTACAAATGAACA GTCTGCAGCCGG
ECAATGGGCAGCCGGAGAACAACTACAA TATTGTGCTCGCECTC
CTGACCATCAGC
GCTTAAGAGCTGA AAGACTTCGCAAC
GACCACGCCTCCCGTGCTGGACTCCGACG ArracmccErGGEt AGTCTGCAGCCG
GGACACTGCCGTC TTATTACTGTCAG
GCTCCTICTTCCIGTACAGCAAGCTCACCG TGGTGCTATGGACTAC
GAAGACTTCGCA
TATTATTGTGCTCG CAATCTICTTATT
TGGACAAGAGCCGCTGGCAGCAGGGGAA TGGGGTCAAGGAACC
ACTTATTACTGTC
CGCGTTTCCGGGT CTCTGATCACGTT
CGTCTTCTCATGCTCCGTGATGCATGAGGC CTGGICACCGTUCCT
AGCAATACTACT
TCTTACCATCCTAT CGGACAGGGTAC
TCTGCACAACCACTACACGCAGAAGAGCC CG
GGCCGATCACET
GGACTACTGGGGT CAAGGIGGAGAT
TCTCCCTGTCTCCGGGTAAAAGCGGCAGC

CAAGGAACCCTGG CAAA
GAGACTCCCGGGACCTCAGAGTCCGCCAC
CCAAGGTGGAG mo n TCACCGTCTCCTCG
ACCCGAAAGIGGIGGCGGA
ATCAAATGA

, t..) S...:1 c tn VI
t coo Alternate Co-Receptor Antibody No. name FZD recognized recognized 2746 Fe FZD6 2747 Fel FZD6 2864 FZD1, FZD2, FZD4, FZD5, FZD7, 2870 FZD1, FZD2, FZD4, FZD5, FZD7, 2876 F2"1 FZD2 2886 F217 FZD2, FZD7(?) 2939 FZD1, FZD2, FZD4, FZD5, FZD7, FZD9, FZD10 2974 F9p3.1 FZD9, FZD10 5019 FP FZD1, FZD2, FZD4, FZD5, FZD7, 5038 F4=1 FZD4 5044 F4.4 FZD4 5048 F4"7 FZD4 5056 FP4 FZD1, FZD2, FZD4, FZD6, FZD7, 5062 F4=2 FZD4 5063 F4.5 FZD4 5067 F4 FZD1, FZD2, FZD4, FZD5, FZD7, 5075 FP5 FZD1, FZD2, FZD4, FZD5, FZD7, 5076 FP7 FZD1, FZD2, FZD4, FZD5, FZD7, 5080 Ft3 FZD4 5081 Fa.6 FZD4 2459 L5i Nomenclature Description 5019-2539 Hi- A bispecific immunoglobulin with a first binding domain for FZD derived from IgG antibody 5019 and a second biding domain for the Wnt3a binding site on LRP6, derived from antibody 2539, wherein both binding domains are on the same side of the Fc domain 5019-2542 Hi- A bispecific immunoglobulin with a first binding domain for FZD derived from IgG antibody 5019 and a second binding domain for the Wirtl binding site on LRP6, derived from antibody 2542, wherein both binding domains are on the same side of the Fc domain 5019-2539-1C/H A bispecific diabody comprising an Fe domain in a knob in hole configuration and (FZD/LRP6-W3) one binding domain comprising a binding site for FZD derived from antibody 5019 and a binding site for the Wnt3a binding site on LRP6 that is derived from antibody 5019-2542-KM A bispecific diabody comprising an Fc domain in a knob in hole configuration and (FZD/LRP6-W1) one binding domain having a binding site for FZD derived from antibody 5019 and a binding site for the Wntl binding site on LRP6 that is derived from antibody 5019-Fc-2539 A tetravalent binding molecule comprising an Fc domain and a binding domain for FZD and a binding domain for LRP6, wherein the binding domain for FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5019, and the binding domain for LRP6 is in a diabody configuration that is bivalent and monspecific for binding the Wnt 3a binding site and derived from antibody 5019-Fc-2542 A tetravalent binding molecule comprising an Fc domain and a binding domain for FZD and a binding domain for LRP6, wherein the binding domain for FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5019, and The binding domain for LRP6 is in a diabody configuration that is bivalent and monspecific for binding the Wnt 1 binding site and derived from antibody 5019Ag A tetravalent binding molecule comprising an Fc domain and a binding domain for 5019-1C/H-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for FZD
is in a diabody configuration that is bivalent, monospecific and derived from antibody 5019, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 5019-K/H-2539- A tetravalent binding molecule comprising an Fc domain and a binding domain for MBP FZD and a binding domain comprising a binding site for LRP6 and a Maltose Binding Protein (MBP), wherein the FC domain is in a knob in hole configuration, the binding domain for FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5019, and the binding domain having a binding site for LRP6 is in a diabody configuration having a binding site for derived from antibody 2539 and a binding site for the Maltose Binding Protein.
5038Ag A tetravalent binding molecule comprising an Fc domain and a binding domain for a 5038-K/H-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific, and derived from antibody 5039, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 5044Ag A tetravalent binding molecule comprising an Fc domain and a binding domain for a 5044-KJH-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5044, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 5048Ag A tetravalent binding molecule comprising an Fc domain and a binding domain for a 5048-IC/11-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 50448, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 5063Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a 5063-1CJH-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5063, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Writl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2890Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a 2890-1C/H-2539- FZD2 and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD2 is in a diabody configuration that is bivalent, monospecific and derived from antibody 2890, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539.
12735Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a 12735-1CM-2539- FZD7 and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 12735, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 5080Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a 5080-1C/H-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5080, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 5081Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for 50814CM-2539- FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole 2542 configuration, the binding domain for FZD
is in a diabody configuration that is bivalent, monospecific and derived from antibody 5081, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2876Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a FZD and a binding domain for LRP6, wherein the Fe domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 2876, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2890Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, monospecific, and derived from antibody 2890, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2886Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent and derived from antibody 2886, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2747Ag A tetravalent binding molecule comprising an Fc domain and a binding domain for a FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, derived from antibody 2747, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2969Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, derived from antibody 2969, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 2974Ag A tetravalent binding molecule comprising an Fe domain and a binding domain for a FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, the binding domain for the FZD is in a diabody configuration that is bivalent, derived from antibody 2074, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 Homodiabody A diabody comprising an Fe domain and two binding sites for the Wnt3a binding 2539-Fe site on LRP6 that is derived from antibody 2539 Homodiabody A diabody comprising an Fe domain and two binding sites for the Wntl binding site 2542-Fe on LRP6 that is derived from antibody FP+P-L61+3 A tetravalent binding molecule comprising an Fe domain and a binding domain for FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, wherein the binding domain for FZD is in a diabody configuration that is bivalent, monospecific and derived from antibody 5019, and the binding domain for LRP6 is in a diabody configuration that is bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 FP*+P*-L61+3 A tetravalent binding molecule comprising an Fe domain and a binding domain for FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, wherein the binding domain for FZD is in a scFV configuration such that the binding domain is bivalent, monospecific, and the scFv is derived from antibody 5019, and the binding domain for LRP6 is in a diabody configuration bivalent and bispecific binding the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and 2539 FP+P-L61*+3* A tetravalent binding molecule comprising an Fe domain and a binding domain for FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, wherein the binding domain for FZD is a diabody configuration that is bivalent and monospecific and derived from antibody 5019, and the binding domain for LRP6 is in a scFv configuration that is bispecific for binding the Wntl and Wnt 3a binding sites on LRP6 and the scFv is derived from antibodies 2542 and FP*+P*461*+3* A tetravalent binding molecule comprising an Fe domain and a binding domain for FZD and a binding domain for LRP6, wherein the FC domain is in a knob in hole configuration, wherein the binding domain for FZD is in a scFv configuration that is bivalent, monospecific, and derived from antibody 5019, and the binding domain for LRP6 is in a scFv configuration that is bivalent and bispecific for binding to the Wntl and Wnt 3a binding sites on LRP6 and is derived from antibodies 2542 and

Claims

We claim

1. A method for activating a Wnt signaling pathway in a cell, said method comprising contacting a cell having a Frizzled2 (FZD2) receptor or Frizzled7 (FZD7) and a Wnt co-receptor with a multivalent binding molecule, wherein the multivalent binding molecule comprises (a) an Fc domain, or fragment thereof comprising a CH3 domain, having a C-terminus and an N-terminus, (b) (i) a FZD2 binding domain having at least two binding sites wherein at least one binding site binds to the FZD2 receptor and comprises a light-chain variable domain (VL) that is 50%, 55%, 60%, 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VL of 2890-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 85) or CDRs of the VL of 2890-hole-2539-2542 , and comprises a heavy-chain variable domain (VH) comprising VH of 2890-hole-2542 (having the amino acid sequence encoded by SEQ ID NO: 84), or CDRs of the VHs of 2890-hole-2539-2542 or 12735-hole-2539-2542, or (ii) a FZD7 binding domain having at least two binding sites wherein at least one binding site binds to the FZD7 receptor and comprises a light-chain variable domain (VL) that is 50%, 55%, 60%, 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a VL of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 87) or CDRs of the VHs of 12735-hole-2539-2542 and comprises a heavy-chain variable domain (VII) comprising VH of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO:
86), or CDRs of the VHs of 12735-hole-2539-2542, and (c) a Wnt co-receptor domain having at least two binding sites wherein at least one binding site binds to the Wnt co-receptor, wherein the FZD2 or FZD7 binding domain is attached to one terminus of the Fc domain or one terminus of the fragment thereof, and the Wnt co-receptor binding domain is attached to the other terminus of the Fc domain or the other terminus of the fragment thereof.

2. The method of claim 1, wherein the FZD2 or FZD7 binding domain comprises, (a)(i) a diabody that binds the FZD2 receptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL and VH of the other peptide thereby forming the diabody, and wherein the VL comprises the VL of 2890-hole-2539-2542(having the amino acid sequence encoded by SEQ ID NO: 85) or CDRs of the VL of 2890-hole-2539-2542 , and the VH comprises the VH of 2890-hole-2542 (having the amino acid sequence encoded by SEQ ID NO: 84), or CDRs of the VH of 2890-hole-2539-2542, or (ii) a diabocly that binds the FZD7 receptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL and VH of the other peptide thereby forming the diabody, and wherein the VL comprises the VL of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 87) or CDRs of the VL of 12735-hole-2539-2542, and the VH comprises the VH of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 86), or CDRs of the VH of 12735-hole-2539-2542, or (b) (i) an scEv comprising a VL comprising a VL of 2890-hole-2539-2542 or (having the amino acid sequence encoded by SEQ ID NO: 85) or CDRs of the VL
of 2890-hole-2539-2542 and a VH region comprising the VH of 2890-hole-2542 (having the amino acid sequence encoded by SEQ ID NO: 84), or CDRs of the VHs of 2890-hole-2539-2542 o, and binds the FZD2 receptor, or (ii) an scFv comprising a VL comprising a VL of 12735-hole-2539-2542 (having an amino acid sequence encoded by SEQ ID NO: 87) or CDRs of the VL of 2890-hole-2539-2542 or 12735-hole-2539-2542 and a VH region comprising the VH of 12735-hole-2539-2542 (having an amino acid sequence encoded by SEQ ID No:
86), or CDRs of the VHs of 12735-hole-2539-2542, and binds the FZD7 receptor and the Wnt co-receptor binding domain comprises (c) a diabody that binds the Wnt co receptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL and VH of the other peptide thereby forming the diabody, or, (de) an scFv comprising VL and VH regions that binds the co-receptor, or (e) an endogenous ligand of the co-receptor or a fragment of such ligand that binds the co-receptor.

3. The method of claim 1, wherein the Wnt co-receptor binding domain binds to a Wnt ligand binding site on the Wnt co-receptor.

4. The method of claim 3, wherein the Wnt co-receptor binding domain binds to Wnt3 and/or Wntl binding sites.

5. The method of claim 1, wherein the Fc domain is an Igo Fc domain.

6. A multivalent binding molecule, wherein the multivalent binding molecule comprises (a) an Fc domain, or fragment thereof comprising a CH3 domain, having a C-terminus and an N-terminus, (b) (i) a FZD2 binding domain having at least two binding sites wherein at least one binding site comprises a light-chain variable domain (VL) comprising VL of hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 85 ) or CDRs of the VL of 2890-hole-2539-2542 , and comprises a heavy-chain variable domain (VH) comprising VH of 2890-hole-2542 (having the amino acid sequence encoded by SEQ ID NO: 84), or CDRs of the VHs of 2890-hole-2539-2542, and binds to the FZD2 receptor, or (ii) a FZD7 binding domain having at least two binding sites wherein at least one binding site comprises a light-chain variable domain (VL) comprising VL 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO:87) or CDRs of the VL of 12735-hole-2539-2542, and comprises a heavy-chain variable domain (VH) comprising VH of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 86), or CDRs of the VHs of 2890-hole-2539-2542 or 12735-hole-2539-2542, and binds to the FZD7 receptor and (c) a Wnt co-receptor binding domain having at least two binding sites wherein at least one binding site binds to the Wnt co-receptor, wherein the FZD2 or FZD7 binding domain is attached to one terminus of the Fc domain and the Wnt co-receptor binding domain is attached to the other terminus of the Fc domain.

7. The multivalent binding molecule of claim 6, wherein the FZD2 or FZD7 binding domain comprises, (a)(i) a diabody that binds the FZD2 receptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL

and VH of the other peptide thereby forming the diabody, and wherein the VL
comprises the VL of 2890-hole-2539-2542 (having an amino acid sequence encoded by SEQ ID NO: 85) or CDRs of the VL of 2890-hole-2539-2542, and the VH
comprises the VH of 2890-hole-2542 (encoded by SEQ ID NO: 84), or CDRs of the VH of 2890-hole-2539-2542, or (ii) a diabody that binds the FZD7 receptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VII) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL and VII of the other peptide thereby forming the diabody, and wherein the VL comprises the VL of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 87) or CDRs of the VL of 12735-hole-2539-2542, and the VH comprises the VH of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 86), or CDRs of the VH of 12735-hole-2539-2542, or (b) (i) an scFv comprising VL and VH regions that bind the FZD2 receptor, wherein the VL comprises the VL of 2890-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 85) or CDRs of the VL of 2890-hole-2539-2542 or 12735-hole-2539-2542, and the VH comprises the VH of 2890-hole-2542 (having the amino acid sequence encoded by SEQ ID NO: 86), or CDRs of the VH of 2890-hole-2539-2542, or (ii) an scFv comprising VL and VH regions that bind the FZD7 receptor, wherein the VL comprises the VL of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 87) or CDRs of the VL of 12735-hole-2539-2542, and the VH comprises the VH of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 86), or CDRs of the VH of 12735-hole-2539-2542, and the Wnt co-receptor binding domain comprises (d) a diabody that binds the coreceptor, said diabody comprising two peptides each peptide comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) wherein the VH and the VL from one peptide pair to the VL

and VH of the other peptide thereby forming the diabody, wherein the VL
comprises the VL of 2890-hole-2539-2542, 2890-knob-2539-2542, 12735-hole-2539-2542 or 12735-knob-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 50 or 52) or CDRs of the VL of 2890-hole-2539-2542 or 12735-hole-2539-2542 or 12735-knob-2539-2542, and the VH comprises the VH of 2890-hole-2542, 2890-knob-2542, 12735-hole-2539-2542 or 12735-knob-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 49 or 53), or CDRs of the VH of 2890-hole-2539-2542 or 12735-hole-2539-2542, or, (e) an scFv comprising VL and VH regions that bind the co-receptor, wherein the VL
comprises the VL of 2890-hole-2539-2542, 2890-knob-2539-2542 or 12735-hole-2539-2542, or 12735-knob-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 50 or 52) or CDRs of the VL of 2890-hole-2539-2542 or 12735-hole-2539-2542, and the VH comprises the VH of 2890-hole-2542, 2890-hole-2539-2542 or 12735-hole-2539-2542 or 12735-knob-2539-2542 (having the amino acid sequence encoded by SEQ ID No: 49 or 53), or CDRs of the VH of 2890-hole-2539-2542 or 12735-hole-2539-2542.

8. The multivalent binding molecule of claim 6, wherein at least one of the binding domains is bispecific.

9. The multivalent binding molecule of claim 6, comprising a first peptide comprising SEQ ID NO: 77 and a second peptide comprises SEQ ID NO: 79 and binds FZD2.

10. The multivalent binding molecule of 6, comprising a first peptide comprising SEQ
ID NO: 81 and a second peptide comprising SEQ ID NO: 83 and binds FZD7.

11. The multivalent binding molecule of claim 6, comprising a first peptide consisting essentially of SEQ ID NO: 77 and a second peptide consisting essentially of SEQ
ID NO: 79 and binds FZD2.

12. The multivalent binding molecule of 6, comprising a first peptide consisting essentially of SEQ ID NO: 81 and a second peptide consisting essentially of SEQ ID NO: 83.

13. A pharmaceutical composition comprising a multivalent binding molecule of any one of claims 6-12 and a pharmaceutically acceptable carrier.

14. A method for enhancing tissue regeneration in a subject in need thereof, or treating a subject having a condition associated with reduced Wnt signaling comprising administering a multivalent binding molecule of any one of claims 6 to 12 to the subject in an amount sufficient to enhance tissue regeneration or alleviate symptoms associated with the condition.

15. The method of claim 14, wherein the tissue is bone tissue or intestinal tissue.

16. A method for facilitating the interaction of a FZD2 or FZD7 receptor and a Wnt co-receptor on a cell thereby activating a Wnt signaling pathway in the cell comprising, a) selecting an Fc domain, or fragment thereof comprising a CH3 domain, having a C-tenninus and an N-terminus b) linking a bivalent FZD2 or FZD7 receptor binding domain comprising the VL that binds the FZD2 receptor of 2890-hole-2539-2542 or the FZD7 receptor of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ 1D NO 85 or 87 respectively) or CDRs of the VL of 2890-hole-2539-2542 or 12735-hole-2539-2542, and the VH comprises the VH of 2890-hole-2542 or 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO: 84 or 86 respectively), or CDRs of the VII of 2890-hole-2539-2542 or 12735-hole-2539-2542, on one terminus of the Fc domain and linking a bivalent Wnt co-receptor binding domain on the other terminus of the Fc domain thereby forming a tetravalent binding molecule;
c) contacting said tetravalent binding molecule with the cell expressing said FZD2 or FZD7 receptor and Wnt co-receptor under conditions wherein the tetravalent binding molecule binds to the FZD2 or FZD7 receptor and the Wnt co-receptor thereby activating the Wnt signaling pathway.

17. The method of claim 16, wherein the bivalent FZD2 or FZD7 receptor binding domain comprises a diabody comprising the VL of 2890-hole-2539-2542 or of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO 85 or 87) or CDRs of the VL of 2890-hole-2539-2542 or of 12735-hole-2539-2542, and the VH comprises the VH
of 2890-hole-2542 or of 12735-hole-2539-2542 (having the amino acid sequence encoded by SEQ ID NO 84 or 86), or CDRs of the VH of 2890-hole-2539-2542 or of 12735-hole-2542 and the bivalent Wnt receptor binding domain comprises a diabody that binds a Wnt co-receptor.

18 The method of claim 17, wherein the diabody that binds a Wnt co-receptor binds to one or both of Wntl or Wnt3a binding sites on the Wnt co-receptor.